IL-8 antibodies

ABSTRACT

One nonexclusive aspect provides molecules further improved from antibodies that can bind to antigens in an ion concentration-dependent manner. An alternative nonexclusive aspect provides safe and more advantageous Fc region variants that have decreased binding to pre-existing ADA. An alternative nonexclusive aspect provides novel IL-8 antibodies that are superior as pharmaceuticals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to and claims priority to Japanese PriorityPatent Applications No. 2015-021371, filed in Japan on Feb. 5, 2015, andNo. 2015-185254, filed in Japan on Sep. 18, 2015. The content of theseapplications is incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

This application includes a “Sequence Listing.ascii.txt,” 823,904 bytes,created on Feb. 3, 2016, and submitted electronically via EFS-Web, whichis hereby incorporated by reference in its entirety.

TECHNICAL FIELD

In one nonexclusive aspect, the disclosure relates to antibodiescomprising an antigen-binding domain whose antigen-binding activitychanges according to the ion concentration condition and pharmacueticcompositions containing the antibodies. Nucleic acids ecoding theseantibodies and host cells containing the nucleic acids are alsoprovided, as are uses and production methods of the antibodies andpharmaceutical compositions. In another nonexclusive aspect, thedisclosure provides Fc region variants and antibodies containing suchvariants and pharmaceutical compositions containing the Fc regionvariants and antibodies. Nucleic acids ecoding the Fc region variantsand antibodies, and host cells containing the nucleic acids are alsoprovided, as are uses and production methods of the Fc region variantsand antibodies and pharmaceutical compositions. In a third non-exclusiveaspect, the disclosure provides anti-IL-8 antibodies, pharmaceuticalcompositions containing the antibodies, nucleic acids encoding theantibodies, and host cells containing the nucleic acids. Productionmethods and uses of the IL-8 antibodies and pharmaceutical compositionin the treatment of for example, IL-8-associated disorders, are alsoprovided.

BACKGROUND

Antibodies attract attention as pharmaceuticals because they are highlystable in plasma and have few side effects. A number of IgG-typetherapeutic antibodies are on the market, and even now many therapeuticantibodies are under development (Reichert et al., Nat. Biotechnol.23:1073-1078 (2005); Pavlou et al., Eur. J. Pharm. Biopharm.59(3):389-396 (2005)). Meanwhile, various techniques are being developedfor second-generation therapeutic antibodies; including technologies forimproving effector function, antigen-binding ability, pharmacokineticsor stability, and reducing the risk of immunogenicity (Kim et al., Mol.Cells. 20 (1):17-29 (2005)). The dosage for therapeutic antibodies isgenerally very high, and consequently the development of therapeuticantibodies confronts issues such as difficulty in producing subcutaneousformulations and high production costs. Methods for improvingtherapeutic antibody pharmacokinetics, pharmacodynamics, and antigenbinding properties provide ways to reduce the dosage and productioncosts associated with therapeutic antibodies.

The substitution of amino acid residues in the constant region providesone method for improving antibody pharmacokinetics (Hinton et al., J.Immunol. 176 (1):346-356 (2006); Ghetie et al., Nat. Biotechnol.15(7):637-640 (1997)). The technique of affinity maturation provides amethod for enhancing antigen-neutralizing ability of an antibody (Rajpalet al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471 (2005); Wu et al.,J. Mol. Biol. 368:652 (2007)), and may increase the antigen-bindingactivity by introducing mutation(s) into amino acid residue(s) in theCDRs and/or framework regions of an antibody variable domain. Improvingthe antigen-binding properties of an antibody may improve the biologicalactivity of the antibody in vitro or reduce the dosage, and may furtherimprove the efficacy in vivo (in the body) (Wu et al., J. Mol. Biol.368:652-665 (2007)).

The amount of antigen that can be neutralized by one antibody moleculedepends on the affinity of the antibody for the antigen; and thus, it ispossible to neutralize an antigen with a small amount of antibody byincreasing affinity. Antibody affinity for an antigen may routinely beincreased using various known methods (see, e.g., Rajpal et al., Proc.Natl. Acad. Sci. USA 102(24):8466-8471 (2005)). Further, it istheoretically possible to neutralize one antigen molecule (2 antigenswhen an antibody is bivalent) with one antibody molecule, if it can bindcovalently to the antigen to make the affinity infinite. Nevertheless,one limitation for therapeutic antibody development thus far is that oneantibody molecule typically only binds to and neutralizes one antigenmolecule (2 antigens when an antibody is bivalent). Recently it has beenreported that the use of an antibody that binds to an antigen in apH-dependent manner (herein below also referred to as “pH-dependentantibody” or “pH-dependent-binding antibody”) enables one antibodymolecule to bind to and neutralize multiple antigen molecules (see,e.g., WO2009/125825; Igawa et al., Nat. Biotechnol. 28:1203-1207(2010)). A pH-dependent antibody binds to an antigen strongly under theneutral pH conditions in the plasma, and dissociates from the antigenunder the acidic pH condition within the endosome of a cell. Afterdissociation from the antigen, the antibody is recycled to the plasma byFcRn and is then free to bind to and neutralize another antigenmolecule; and thus one pH-dependent antibody may repeatedly bind to andneutralize multiple antigen molecules.

It has recently been reported that antibody recycling properties can beachieved by focusing on the difference of calcium (Ca) ion concentrationbetween plasma and endosome, and using an antibody with anantigen-antibody interaction that demonstrates calcium dependency(herein below also referred to as “calcium ion concentration-dependentantibody”)(WO2012/073992). (Herein below, a pH-dependent antibody and a“calcium ion concentration-dependent antibody” are collectively referredto as a “pH/Ca concentration-dependent antibody”.)

By binding to FcRn, IgG antibodies have long retention in plasma. Thebinding between an IgG antibody and FcRn is strong under an acidic pHconditions (for example, pH 5.8), but there is almost no binding under aneutral pH condition (for example, pH 7.4). An IgG antibody is taken upinto cells non-specifically, and returned to cell surface by binding toFcRn in the endosome under the acidic pH conditions in the endosome. TheIgG then dissociates from the FcRn under the neutral pH conditions inthe plasma.

It is reported that a pH-dependent antibody that has been modified toincrease its FcRn binding under neutral pH conditions has the ability torepeatedly bind to and eliminate antigen molecules from plasma; and thusadministration of such an antibody allows antigen elimination fromplasma (WO2011/122011). According to this report, a pH-dependentantibody that has been modified to increase its FcRn binding underneutral pH conditions (for example, pH 7.4) can further accelerate theelimination of the antigen compared to a pH-dependent antibody thatcomprises the Fc region of a native IgG antibody (WO2011/122011).

Meanwhile, when mutations are introduced into the Fc region of an IgGantibody to eliminate its binding to FcRn under acidic pH conditions, itcan no longer be recycled from the endosome into the plasma, whichsignificantly compromises the antibody's retention in the plasma. Withthat, a method of increasing FcRn binding under acidic pH conditions isreported as a method for improving the plasma retention of an IgGantibody. Introducing amino acid modifications into the Fc region of anIgG antibody to increase its FcRn binding under acidic pH conditions canenhance the efficacy of recycling from the endosome to plasma, which asa result leads to an improvement in plasma retention. For instance, themodifications M252Y/S254T/T256E (YTE; Dall'Acqua et al., J. Biol. Chem.281:23514-235249 (2006)), M428L/N434S (LS; Zalevsky et al., Nat.Biotechnol. 28:157-159 (2010)), and N434H (Zheng et al., Clin. Pharm. &Ther. 89(2):283-290 (2011)), have been reported to result in increasedantibody half-life relative to native IgG1.

However, in addition to the concern that the immunogenicity oroccurrence rate of aggregates may worsen in an antibody that comprisessuch an Fc region variant whose FcRn binding is increased under aneutral pH condition or an acidic pH condition, an increase in thebinding against an anti-drug antibody (herein below also referred to as“Pre-existing ADA”) (for example, rheumatoid factor) present in apatient before administration of a therapeutic antibody has been furtherreported (WO2013/046722, WO2013/046704). WO2013/046704 reports that anFc region variant containing specific mutations (represented by tworesidue modifications of Q438R/S440E according to EU numbering) increasethe binding to FcRn under acidic pH conditions and also showed asignificant reduction in binding to rheumatoid factor compared tounmodified native Fc. However, WO2013/046704 does not specificallydemonstrate that this Fc region variant has superior plasma retention toan antibody with native Fc region.

Accordingly, safe and more favorable Fc region variants with furtherimproved plasma retention that do not show binding to pre-existing ADAare desired.

Antibody-dependent cellular cytotoxicity (herein below noted as “ADCC”),complement-dependent cytotoxicity (herein below noted as “CDC”),antibody-dependent cellular phagocytosis (ADCP) which is phagocytosis oftarget cells mediated by an IgG antibody are reported as effectorfunctions of an IgG antibody. In order for an IgG antibody to mediateADCC activity or ADCP activity, the Fc region of the IgG antibody mustbind to an antibody receptor present on the surface of an effector cellsuch as a killer cell, natural killer cell or activated macrophage(noted as “Fcγreceptor”, “FcgR”, “Fc gamma receptor” or “FcγR” withinthe scope of Disclosure A described herein). In human, FcγRIa, FcγRIIa,FcγRIIb, FcγRIIIa and FcγRIIIb isoforms are reported as FcγR familyproteins, and their respective allotypes have also been reported(Jefferis et al., Immunol. Lett. 82:57-65 (2002)). The balance of therespective affinity of an antibody for an activating receptor comprisingFcγRIa, FcγRIIa, FcγRIIIa or FcγRIIIb, and an inhibitory receptorcomprising FcγRIIb is an important element in optimizing the antibodyeffector functions.

Various techniques that increase or improve the activity of atherapeutic antibody against an antigen have been reported so far. Forinstance, the activity of an antibody to bind to an activating FcγR(s)plays an important role in the cytotoxicity of the antibody, andconsequently, antibodies that target a membrane-type antigen and thathave increased cytotoxicity resulting from enhanced activating FcγR(s)binding have been developed. See, e.g., WO2000/042072; WO2006/019447;Lazar et al., Proc. Nat. Acad. Sci. USA. 103:4005-4010 (2006); Shinkawaet al., J. Biol. Chem. 278, 3466-3473 (2003); Clynes et al., Proc. Natl.Acad. Sci. USA 95:652-656 (1998); Clynes et al., Nat. Med. 6:443-446(2000)). Similarly, the binding activity towards an inhibitory FcγR(FcγRIIb in human) plays an important role in the immunosuppressiveactivity, agonist activity, and thus there has been research onantibodies with increased inhibitory FcγR-binding activity that target amembrane-type antigen (Li et al., Proc. Nat. Acad. Sci. USA. 109(27):10966-10971 (2012)). Further, the influence of FcγR binding of anantibody that binds to a soluble antigen has been examined mainly fromthe viewpoint of side effects (Scappaticci et al., J. Natl. Cancer Inst.99 (16):1232-1239 (2007)). For instance, when an antibody with increasedFcγRIIb binding is used as a drug, one can expect reduced risk from thegeneration of anti-drug antibodies (Desai et al., J. Immunol.178(10):6217-6226 (2007)).

More recently, it has been reported that introducing amino acidmodifications into the Fc region of an IgG antibody to increase theactivity of an antibody that targets a soluble antigen to bind to anactivating and/or inhibitory FcγR(s) can further accelerate eliminationof the antigen from serum (WO2012/115241, WO2013/047752, WO2013/125667,WO2014/030728). Also, an Fc region variant has been identified, whichshows almost no change in its FcγRIIb-binding activity from a native IgGantibody Fc region, but has reduced activity to other activating FcγRs(WO2014/163101).

The plasma retention of a soluble antigen is very short compared to anantibody that has an FcRn-mediated recycling mechanism, and thus asoluble antigen may display increased plasma retention and plasmaconcentration by binding to an antibody that has such a recyclingmechanism (for example, an antibody that does not have thecharacteristics of a pH/Ca concentration-dependent antibody).Accordingly, for example, when a soluble antigen in plasma has multipletypes of physiological functions, even if one type of physiologicalfunctions is blocked as a result of antibody binding, the plasmaconcentration of the antigen may worsen the pathogenic symptoms causedby the other physiological functions as a result of the increased plasmaretention and/or plasma concentration of the antigen resulting from theantibody binding. In this case, in addition to a method of applying theabove-mentioned exemplified modifications to an antibody to accelerateantigen elimination, for example, a method of utilizing the formation ofa multivalent immune complex from multiple pH/Ca concentration-dependentantibodies and multiple antigens, and increasing the binding to FcRn,FcγR(s), a complement receptor, has been reported (WO2013/081143).

Even when the Fc region is not modified, it is reported that bymodifying amino acid residue(s) so as to change the charge of such aminoacid residue(s) which may be exposed on the surface of an antibodyvariable region to increase or decrease the isoelectric point (pI) ofthe antibody, it is possible to control the half-life of the antibody inblood regardless of the type of target antigen or antibody, and withoutsubstantially reducing the antigen-binding activity of the antibody(WO2007/114319: techniques of substituting amino acids mainly in the FR;WO2009/041643: techniques of substituting amino acids mainly in CDR).These documents show that it is possible to prolong the plasma half-lifeof an antibody by reducing the antibody's pI, and conversely shorten theplasma half-life of an antibody by increasing the antibody's pI.

With regard to modification of the charge of amino acid residues in theconstant region of an antibody, it has been reported that the uptake ofan antigen into cells can be promoted by modifying the charge ofspecific amino acid residue(s), particularly in its CH3 domain, toincrease the antibody's pI, and it is also described that thismodification preferably does not interfere with the binding to FcRn(WO2014/145159). It has also been reported that modifying the charge ofamino acid residues in the constant region (mainly CH1 domain) of anantibody to reduce pI can prolong the half-life of the antibody inplasma, and in combination with mutations of amino acid residues toincrease FcRn binding, can enhance its binding to FcRn and prolong theplasma half-life of the antibody (WO2012/016227).

Meanwhile, when such modification techniques designed for increasing orreducing the pI of an antibody are combined with techniques other thanthe modification technique to increase or reduce the binding to FcRn orFcγR(s), it is unclear whether there is an effect in promoting theplasma retention of the antibody or elimination of the antigen fromplasma.

The extracellular matrix (ECM) is a structure that covers cells in vivo,and is mainly constituted by glycoproteins such as collagen,proteoglycan, fibronectin, and laminin. The role of the ECM in vivo isto create a microenvironment for cells to survive, and the ECM isimportant in various functions carried out by cells such as, cellproliferation and cell adhesion.

The ECM has been reported to be involved in the in vivo kinetics ofproteins administered to a living body. Blood concentration of theVEGF-Trap molecule, which is a fusion protein between the VEGF receptorand Fc, when subcutaneously administered was examined (Holash et al.,Proc. Natl. Acad. Sci., 99(17):11393-11398 (2002)). Plasma concentrationof the subcutaneously administered VEGF-Trap molecule which has a highpI, was low, and therefore its bioavailability was low. A modifiedVEGF-Trap molecule whose pI was reduced by amino acid substitutions hasa higher plasma concentration, and its bioavailability could beimproved. Further, change in the bioavailability correlates with thestrength of binding to the ECM, and thus it became evident that thebioavailability of the VEGF-Trap molecule when subcutaneouslyadministered depends on the strength of its binding to the ECM at thesubcutaneous site.

WO2012/093704 reports that there is an inverse correlation betweenantibody binding to the ECM and plasma retention, and consequently,antibody molecules that do not bind to the ECM have better plasmaretention when compared to antibodies that bind to the ECM.

As such, techniques for reducing extracellular matrix binding with theobjective of improving protein bioavailability in vivo and plasmaretention have been reported. By contrast, the advantages of increasingantibody binding to the ECM have not been identified so far.

Human IL-8 (Interleukin 8) is a chemokine family member that is 72 or 77amino acid residues in length. The term “chemokine” is a collective termfor a family of proteins with a molecular weight of 8-12 kDa and contain4 cysteine residues that form intermolecular disulfide bonds. Chemokinesare categorized into CC chemokine, CXC chemokine, C chemokine, CA3Cchemokine according to the characteristics of the cysteine arrangement.IL-8 is classified as a CXC chemokine, and is also referred to as CXCL8.

IL-8 exists in solution in monomeric and homodimeric form. The IL-8monomer contains antiparallel β sheets, and has a structure in which aC-terminal a helix traverses and covers the β sheets. An IL-8 monomer,in the case of the 72 amino acid form of IL-8, comprises two disulfidecrosslinks between cysteine 7 and cysteine 34, and between cysteine 9and cysteine 50. IL-8 homodimers are stabilized by noncovalentinteractions between the β sheets of the two monomers, as there is nocovalent binding between molecules in homodimers.

IL-8 expression is induced in various cells such as peripheral bloodmonocytes, tissue macrophages, NK cells, fibroblasts, and vascularendothelial cells in response to stimulation by inflammatory cytokines(Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014)).

Chemokines are generally not detectable, or only weakly detectable, innormal tissue, but are strongly detected at inflamed sites, and areinvolved in eliciting inflammation by facilitating infiltration ofleukocyte into inflamed tissue sites. IL-8 is a proinflammatorychemokine that is known to activate neutrophils, promote expression ofcell adhesion molecules, and enhance neutrophil adhesion to vascularendothelial cells. IL-8 also has neutrophil chemotactic capacity andIL-8 produced at a damaged tissue facilitates chemotaxis of neutrophilsadhered to vascular endothelial cells into the tissue, and inducesinflammation along with neutrophil infiltration. IL-8 is also known tobe a potent angiogenic factor for endothelial cells and is involved inpromoting tumor angiogenesis.

Inflammatory diseases associated with elevated (e.g., excess) IL-8levels include, inflammatory diseases of the skin such as inflammatorykeratosis (e.g., psoriasis), atopic dermatitis, contact dermatitis;chronic inflammatory disorders which are autoimmune diseases, such asrheumatoid arthritis, systemic lupus erythematosus (SLE), and Behcet'sdisease; inflammatory bowel diseases such as Crohn's disease andulcerative colitis; inflammatory liver diseases such as hepatitis B,hepatitis C, alcoholic hepatitis, drug-induced allergic hepatitis;inflammatory renal diseases such as glomerulonephritis; inflammatoryrespiratory diseases such as bronchitis and asthma; inflammatory chronicvascular diseases such as atherosclerosis; multiple sclerosis, oralulcer, chorditis, and inflammation associated with using artificialorgans and/or artificial blood vessels. Elevated (e.g., excess) IL-8levels are also associated with malignant tumors such as ovarian cancer,lung cancer, prostate cancer, stomach cancer, breast cancer, melanoma,head and neck cancers, and kidney cancer; sepsis due to infection;cystic fibrosis; and pulmonary fibrosis. (See, e.g., Russo et al., Exp.Rev. Clin. Immunol. 10(5):593-619 (2014), which is herein incorporatedby reference in its entirety).

For several of these diseases, human anti-IL-8 antibodies with highaffinity have been developed as pharmaceutical compositions (Desai etal., J. Immunol. 178(10):6217-6226 (2007)), however, they have not beenlaunched yet. So far, only one pharmaceutical composition comprisingIL-8 antibody is available, which is a murin anti-IL-8 antibody forpsoriasis as external medicine. New anti-IL-8 antibodies for treatmentdiseases are expected.

BRIEF SUMMARY

In one nonexclusive aspect, a non-limited objective of embodiments ofDisclosure A is to provide molecules with improved pharmacokineticproperties over antibodies, such as ion concentration-dependent antigenbinding properties that improve antibody half-life and/or antigenclearance from the plasma.

In one nonexclusive aspect, a non-limited objective of embodiments ofDisclosure B is to provide, safe and more favorable Fc region variantsthat have increased half-life and decreased binding to pre-existinganti-drug antibodies (ADAs).

In one nonexclusive aspect, a non-limited objective of embodiments ofDisclosure C is to provide anti-IL-8 antibodies that have pH-dependentbinding affinity towards IL-8. An additional embodiment relates toanti-IL-8 antibodies that have an effect of rapidly eliminating IL-8compared to a reference antibody when administered to an individual. Inanother embodiment, Disclosure C relates to anti-IL-8 antibodies thatcan stably maintain their IL-8-neutralizing activity when administeredto an individual. In some embodiments, the anti-IL-8 antibodies displayreduced immunogenicity. In additional embodiments, Disclosure C relatesto a method of producing and using the above-mentioned anti-IL-8antibodies. Another alternative non-limited objective of Disclosure Cis, to provide novel anti-IL-8 antibodies that can be included in apharmaceutical composition.

In one nonexclusive aspect, within the scope of Disclosure A as providedherein, the inventors have surprisingly discovered that the ability ofan ion concentration-dependent antibody (which is an antibody comprisingan ion concentration-dependent antigen-binding domain (“anantigen-binding domain whose antigen-binding activity changes accordingto ion concentration conditions”)) to eliminate antigen from plasma canbe accelerated by modifying at least one of the amino acid residuesexposed on the surface of the antibody to increase its isoelectric point(pI). In another nonexclusive aspect, the inventors discovered that anion concentration-dependent antibody with increased pI can furtherincrease the extracellular matrix-binding of the antibody. Thus, withoutbeing confined to a particular theory, the inventors have discoveredthat antigen elimination from plasma can be increased, by increasing thebinding of the antibody towards extracellular matrix.

In one nonexclusive aspect, within the scope of Disclosure B as providedherein, the inventors conducted dedicated research on safe and morefavorable Fc region variants that do not show binding to anti-drugantibodies (pre-existing ADA) and that can further improve plasmaretention. As a result, the inventors have surprisingly discovered thatFc region variants comprising a substitution of position 434 amino acidaccording to EU numbering with Ala (A) and two specific residuemutations (represented by Q438R/S440E according to EU numbering) as acombination of amino acid residue mutations, are preferred formaintaining significant reduction in the binding to rheumatoid factor,along with achieving a plasma retention of an antibody.

In one nonexclusive aspect, within the scope of Disclosure C as providedherein, the inventors generated a number of pH-dependent anti-IL-8antibodies (anti-IL-8 antibodies that bind to IL-8 in a pH-dependentmanner). From the results of various validations, the inventorsidentified pH-dependent anti-IL-8 antibodies that have an effect ofrapidly eliminating IL-8 compared to a reference antibody whenadministered to an individual. In some embodiments the Disclosure Crelates to pH-dependent anti-IL-8 antibodies that can stably maintaintheir IL-8-neutralizing activity. In additional nonlimiting embodiments,the pH-dependent anti-IL-8 antibodies have reduced immunogenicity andexcellent expression levels.

Further, within the scope of Disclosure C, the inventors successfullyobtained anti-IL-8 antibodies that comprise an Fc region whoseFcRn-binding affinity at acidic pH is increased relative to theFcRn-binding affinity of a native Fc region. In an alternative aspect,the inventors successfully obtained anti-IL-8 antibodies that comprisean Fc region whose binding affinity towards pre-existing ADA is reducedrelative to the binding affinity of a native Fc region for thepre-existing ADA. In an alternative aspect, the inventors successfullyobtained anti-IL-8 antibodies comprising an Fc region that whose plasmahalf-life is increased relative to the plasma half-life of a native Fcregion. In an alternative aspect, the inventors successfully obtainedpH-dependent anti-IL-8 antibodies that comprise an Fc region whosebinding affinity towards effector receptors is reduced relative to thebinding affinity of a naturally occurring Fc region for the effectorreceptors. In a different aspect, the inventors identified nucleic acidsencoding the above-mentioned anti-IL-8 antibodies. In another aspect,the inventors also obtained hosts comprising the above-mentioned nucleicacids. In another aspect, the inventors developed a method for producingthe above-mentioned anti-IL-8 antibodies, which comprises culturing theabove-mentioned host. In another aspect, the inventors developed amethod for facilitating the elimination of IL-8 from an individualrelative to a reference antibody, which comprises administering theabove-mentioned anti-IL-8 antibodies to the individual.

In one embodiment, Disclosure A, relates without limitation to,

-   [1] an antibody comprising an antigen-binding domain whose    antigen-binding activity changes according to ion concentration    conditions, wherein its isoelectric point (pI) is increased by the    modification of at least one amino acid residue that may be exposed    on the surface of the antibody;-   [2] the antibody of [1], wherein the antigen is a soluble antigen;-   [3] the antibody of [1] or [2], wherein the antigen-binding domain    is a domain whose antigen-binding activity under a high ion    concentration condition is higher than that under a low ion    concentration condition;-   [4] the antibody of any one of [1] to [3], wherein the ion    concentration is a hydrogen ion concentration (pH) or a calcium ion    concentration;-   [5] the antibody of [4], wherein the ratio of its KD in an acidic pH    range to that in a neutral pH range, KD (acidic pH range)/KD    (neutral pH range), for the antigen, is 2 or higher;-   [6] the antibody of any one of [1] to [5], wherein in the    antigen-binding domain, at least one amino acid residue is    substituted with histidine, or at least one histidine is inserted;-   [7] the antibody of any one of [1] to [6], which can promote    elimination of the antigen from plasma as compared to an antibody    before the modification;-   [8] the antibody of any one of [1] to [7], wherein its extracellular    matrix-binding activity is enhanced as compared to an antibody    before the modification;-   [9] the antibody of any one of [1] to [8], wherein the amino acid    residue modification is amino acid residue substitution;-   [10] the antibody of any one of [1] to [9], wherein the amino acid    residue modification is selected from the group consisting of:    -   (a) substitution of a negatively charged amino acid residue with        an uncharged amino acid residue;    -   (b) substitution of a negatively charged amino acid residue with        a positively charged amino acid residue; and    -   (c) substitution of an uncharged amino acid residue with a        positively charged amino acid residue;-   [11] the antibody of any one of [1] to [10], wherein the antibody    comprises a variable region and/or a constant region, and the amino    acid residue modification is amino acid residue modification in the    variable region and/or the constant region;-   [12] the antibody of [11], wherein the variable region comprises    complementarity-determining region(s) (CDR(s)) and/or framework    region(s) (FR(s));-   [13] the antibody of [12], wherein the variable region comprises a    heavy chain variable region and/or a light chain variable region,    and at least one amino acid residue is modified in a position in a    CDR or a FR selected from the group consisting of:    -   (a) position 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26,        39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a,        82b, 83, 84, 85, 86, 105, 108, 110, and 112 in a FR of the heavy        chain variable region;    -   (b) position 31, 61, 62, 63, 64, 65, and 97 in a CDR of the        heavy chain variable region;    -   (c) position 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38,        39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74,        76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in a FR        of the light chain variable region; and    -   (d) position 24, 25, 26, 27, 52, 53, 54, 55, and 56 in a CDR of        the light chain variable region, according to Kabat numbering;-   [14] the antibody of [13], wherein at least one amino acid residue    is modified in a position in a CDR or a FR selected from the group    consisting of:    -   (a) position 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77,        82, 82a, 82b, 83, 84, 85, and 105 in a FR of the heavy chain        variable region;    -   (b) position 31, 61, 62, 63, 64, 65, and 97 in a CDR of the        heavy chain variable region;    -   (c) position 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and        107 in a FR of the light chain variable region; and    -   (d) position 24, 25, 26, 27, 52, 53, 54, 55, and 56 in a CDR of        the light chain variable region;-   [15] the antibody of any one of [11] to [14], wherein at least one    amino acid residue is modified in a position in the constant region    selected from the group consisting of position 196, 253, 254, 256,    258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330,    342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386,    387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430,    433, 434, and 443, according to EU numbering;-   [16] the antibody of [15], wherein at least one amino acid residue    is modified in a position in the constant region selected from the    group consisting of position 254, 258, 281, 282, 285, 309, 311, 315,    327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 384, 385, 386,    387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434, and 443;-   [17] the antibody of [16], wherein at least one amino acid residue    is modified in a position in the constant region selected from the    group consisting of position 282, 309, 311, 315, 342, 343, 384, 399,    401, 402, and 413, according to EU numbering;-   [18] the antibody of any one of [1] to [17], wherein the constant    region has Fc gamma receptor (FcγR)-binding activity, and wherein    the FcγR-binding activity under a neutral pH condition is enhanced    as compared to that of a reference antibody comprising a constant    region of a native IgG;-   [19] the antibody of [18], wherein the FcγR is FcγRIIb;-   [20] the antibody of any one of [1] to [17], wherein the constant    region has binding activity towards one or more activating FcγR    selected from the group consisting of FcγRIa, FcγRIb, FcγRIc,    FcγRIIIa, FcγRIIIb and FcγRIIa, and towards FcγRIIb, and the    FcγRIIb-binding activity is maintained or enhanced and the binding    activity to the activating FcγRs is decreased, as compared to those    of a reference antibody which differs only in that its constant    region is that of a native IgG;-   [21] the antibody of any one of [1] to [20], wherein the constant    region has FcRn-binding activity, and wherein the FcRn-binding    activity under a neutral pH condition (e.g., pH 7.4) is enhanced as    compared to that of a reference antibody which differs only in that    its constant region is that of a native IgG;-   [22] the antibody of any one of [1] to [21], which is a    multispecific antibody that binds to at least two antigens;-   [23] the antibody of any one of [1] to [22], wherein the antibody is    an IgG antibody;-   [24] a pharmaceutical composition comprising the antibody of any one    of [1] to [23];-   [25] the pharmaceutical composition of [24], which is for promoting    the elimination of an antigen from plasma;-   [26] the pharmaceutical composition of [24] or [25], which is for    enhancing the antibody binding to an extracellular matrix;-   [27] a nucleic acid encoding the antibody of any one of [1] to [23];-   [28] a vector comprising the nucleic acid of [27];-   [29] a host cell comprising the vector of [28];-   [30] a method for producing an antibody comprising an    antigen-binding domain whose antigen-binding activity changes    according to ion concentration conditions, wherein the method    comprises culturing the host cell of [29] and collecting the    antibody from the cell culture;-   [30A] a method for producing an antibody comprising an    antigen-binding domain whose antigen-binding activity changes    according to ion concentration conditions, wherein the method    comprises modifying at least one amino acid residue that may be    exposed on the surface of the antibody so as to increase the    isoelectric point (pI);-   [30B] the method of [30A], wherein at least one amino acid residue    is modified    -   (I) in a position in a CDR or FR selected from the group        consisting of: (a) position 1, 3, 5, 8, 10, 12, 13, 15, 16, 18,        19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76,        77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in        a FR of the heavy chain variable region; (b) position 31, 61,        62, 63, 64, 65, and 97 in a CDR of the heavy chain variable        region; (c) position 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22,        37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69,        70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108        in a FR of the light chain variable region; and (d) position 24,        25, 26, 27, 52, 53, 54, 55, and 56 in a CDR of the light chain        variable region, according to Kabat numbering; or    -   (II) in a position in a constant region selected from the group        consisting of position 196, 253, 254, 256, 258, 278, 280, 281,        282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356,        358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400,        401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443,        according to EU numbering;-   [31] the method of [30A] or [30B], wherein the amino acid residue    modification comprises a modification selected from the group    consisting of:    -   (a) substitution of a negatively charged amino acid residue with        an uncharged amino acid residue;    -   (b) substitution of a negatively charged amino acid residue with        a positively charged amino acid residue;    -   (c) substitution of an uncharged amino acid residue with a        positively charged amino acid residue; and    -   (d) substitution or insertion with histidine in a CDR or FR.-   [32] the method of any one of [30], or [30A] to [30C] which further    optionally comprises any one or more of:    -   (a) selecting an antibody which can promote elimination of an        antigen from plasma;    -   (b) selecting an antibody with enhanced binding activity to an        extracellular matrix;    -   (c) selecting an antibody with enhanced FcγR-binding activity        under a neutral pH condition (e.g., pH 7.4);    -   (d) selecting an antibody with enhanced FcγRIIb-binding activity        under a neutral pH condition (e.g., pH 7.4);    -   (e) selecting an antibody with maintained or enhanced        FcγRIIb-binding activity and decreased binding activity to one        or more activating FcγR, preferably selected from the group        consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and        FcγRIIa;    -   (f) selecting an antibody with enhanced FcRn-binding activity        under a neutral pH condition (e.g., pH 7.4);    -   (g) selecting an antibody with an increased isoelectric point        (pI);    -   (h) confirming the isoelectric point (pI) of the collected        antibody, and then selecting an antibody with an increased        isoelectric point (pI); and    -   (i) selecting an antibody whose antigen-binding activity is        changed or increased according to ion concentration conditions;        -   as compared to a reference antibody;

In an alternative embodiment, Disclosure A relates without limitationto:

-   [A1] an antibody having a constant region, wherein at least one    amino acid residue selected from the group of modification sites    identical to the modification sites in the group defined in [15] or    [16] is modified in the constant region;-   [A2] the antibody of [A1], which further has a heavy-chain variable    region and/or a light-chain variable region, wherein the variable    region has CDR(s) and/or FR(s), and wherein at least one amino acid    residue selected from the group of modification sites identical to    the modification sites in the group defined in [13] or [14] is    modified in a CDR and/or a FR;-   [A3] an antibody having a constant region, wherein at least one    amino acid residue selected from the group of modification sites    identical to the modification sites in the group defined in [15] or    [16] is modified in the constant region so as to increase its pI;-   [A4] the antibody of [A3], which further has a heavy-chain variable    region and/or a light-chain variable region, wherein the variable    region has CDR(s) and/or FR(s), and wherein at least one amino acid    residue selected from the group of modification sites identical to    the modification sites in the group defined in [13] or [14] is    modified in a CDR and/or a FR;-   [A5] an antibody comprising an antigen-binding domain whose    antigen-binding activity changes according to ion concentration    conditions, wherein the antibody has a constant region, and wherein    at least one amino acid residue selected from the group of    modification sites identical to the modification sites in the group    defined in [15] or [16] is modified in the constant region;-   [A6] the antibody of [A5], which further has a heavy-chain variable    region and/or a light-chain variable region, wherein the variable    region has CDR(s) and/or FR(s), and wherein at least one amino acid    residue selected from the group of modification sites identical to    the modification sites in the group defined in [13] or [14] is    modified in a CDR and/or a FR;-   [A7] use of the antibody of any one of [1] to [23] and [A1] to [A6]    in the manufacture of a medicament for promoting antigen elimination    from plasma;-   [A8] use of the antibody of any one of [1] to [23] and [A1] to [A6]    in the manufacture of a medicament for increasing extracellular    matrix binding;-   [A9] use of the antibody of any one of [1] to [23] and [A1] to [A6]    for eliminating an antigen from plasma; and-   [A10] use of the antibody of any one of [1] to [23] and [A1] to [A6]    for increasing extracellular matrix binding.-   [A11] an antibody obtained by the method of any one of [30], [30A],    [30B], [31], [32].

According to various embodiments, Disclosure A encompasses combinationsof one or multiple elements described in any of [1] to [30], [30A],[30B], [31], [32] and [A1] to [A11] mentioned above, in part or as awhole, as long as such a combination is not technically inconsistentwith the common technical knowledge in the art. For example, in someembodiments, Disclosure A encompasses a method for producing a modifiedantibody comprising an antigen-binding domain which promotes eliminationof an antigen from plasma as compared to that before the antibodymodification, wherein the method comprises:

-   -   (a) modifying at least one amino acid residue that may be        exposed on the surface of an antibody, which is:        -   (I) in a position in a CDR or FR selected from the group            consisting of: (a) position 1, 3, 5, 8, 10, 12, 13, 15, 16,            18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73,            75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110,            and 112 in a FR of the heavy chain variable region; (b)            position 31, 61, 62, 63, 64, 65, and 97 in a CDR of the            heavy chain variable region; (c) position 1, 3, 7, 8, 9, 11,            12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49,            57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85,            100, 103, 105, 106, 107, and 108 in a FR of the light chain            variable region; and (d) position 24, 25, 26, 27, 52, 53,            54, 55, and 56 in a CDR of the light chain variable region,            according to Kabat numbering; or        -   (II) in a position in a constant region selected from the            group consisting of position 196, 253, 254, 256, 258, 278,            280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342,            343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386,            387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424,            430, 433, 434, and 443, according to EU numbering;    -   (b) modifying the antigen-binding domain in a way such that the        resulting antigen-binding activity changes according to ion        concentration conditions, wherein said (a) and (b) can be        carried out simultaneously or sequentially;    -   (c) culturing a host cell to express the nucleic acid encoding        the modified antibody; and    -   (d) collecting the modified antibody from the host cell culture.

In further embodiments, the method optionally further comprises any oneor more of:

-   -   (e) selecting an antibody which can promote elimination of an        antigen from plasma;    -   (f) selecting an antibody with enhanced binding activity to an        extracellular matrix;    -   (g) selecting an antibody with enhanced FcγR-binding activity        under a neutral pH condition (e.g., pH 7.4);    -   (h) selecting an antibody with enhanced FcγRIIb-binding activity        under a neutral pH condition (e.g., pH 7.4);    -   (i) selecting an antibody with maintained or enhanced        FcγRIIb-binding activity and decreased binding activity to one        or more activating FcγR, preferably selected from the group        consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and        FcγRIIa;    -   (j) selecting an antibody with enhanced FcRn-binding activity        under a neutral pH condition (e.g., pH 7.4);    -   (k) selecting an antibody with an increased isoelectric point        (pI);    -   (l) confirming the isoelectric point (pI) of the collected        antibody, and then selecting an antibody with an increased        isoelectric point (pI); and    -   (m) selecting an antibody whose antigen-binding activity is        changed or increased according to ion concentration conditions;        -   as compared to the antibody before the modification.

Another embodiment of Disclosure A relates to, for example, withoutlimitation:

-   [D1] a method for producing a modified antibody, whose half-life in    plasma is prolonged or reduced, as compared to that before the    modification of the antibody, wherein the method comprises:    -   (a) modifying a nucleic acid encoding the antibody before the        modification to change the charge of at least one amino acid        residue at a position selected from the group consisting of        position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286,        307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361,        362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413,        415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EU        numbering;    -   (b) culturing a host cell to express the nucleic acid; and    -   (c) collecting the antibody from the host cell culture; or-   [D2] a method for prolonging or reducing the half-life of an    antibody in plasma wherein the method comprises modifying at least    one amino acid residue at a position selected from the group    consisting of position 196, 253, 254, 256, 258, 278, 280, 281, 282,    285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358,    359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401,    402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according    to EU numbering.

In one embodiment, Disclosure B relates to, for example, withoutlimitation:

-   [33] an Fc region variant comprising an FcRn-binding domain, wherein    the FcRn-binding domain comprises Ala at position 434; Glu, Arg,    Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440,    according to EU numbering;-   [34] the Fc region variant of [33], wherein the FcRn-binding domain    comprises Ala at position 434; Arg or Lys at position 438; and Glu    or Asp at position 440, according to EU numbering;-   [35] the Fc region variant of [33] or [34], wherein the FcRn-binding    domain further comprises Ile or Leu at position 428; and/or Ile,    Leu, Val, Thr, or Phe at position 436, according to EU numbering;-   [36] the Fc region variant of [35], wherein the FcRn-binding domain    comprises Leu at position 428; and/or Val or Thr at position 436,    according to EU numbering;-   [37] the Fc region variant of any one of [33] to [36], wherein the    FcRn-binding domain comprises a combination of amino acid    substitutions selected from the group consisting of:    N434A/Q438R/S440E; N434A/Q438R/S440D; N434A/Q438K/S440E;    N434A/Q438K/S440D; N434A/Y436T/Q438R/S440E; N434A/Y436T/Q438R/S440D;    N434A/Y436T/Q438K/S440E; N434A/Y436T/Q438K/S440D;    N434A/Y436V/Q438R/S440E; N434A/Y436V/Q438R/S440D;    N434A/Y436V/Q438K/S440E; N434A/Y436V/Q438K/S440D;    N434A/R435H/F436T/Q438R/S440E; N434A/R435H/F436T/Q438R/S440D;    N434A/R435H/F436T/Q438K/S440E; N434A/R435H/F436T/Q438K/S440D;    N434A/R435H/F436V/Q438R/S440E; N434A/R435H/F436V/Q438R/S440D;    N434A/R435H/F436V/Q438K/S440E; N434A/R435H/F436V/Q438K/S440D;    M428L/N434A/Q438R/S440E; M428L/N434A/Q438R/S440D;    M428L/N434A/Q438K/S440E; M428L/N434A/Q438K/S440D;    M428L/N434A/Y436T/Q438R/S440E; M428L/N434A/Y436T/Q438R/S440D;    M428L/N434A/Y436T/Q438K/S440E; M428L/N434A/Y436T/Q438K/S440D;    M428L/N434A/Y436V/Q438R/S440E; M428L/N434A/Y436V/Q438R/S440D;    M428L/N434A/Y436V/Q438K/S440E; M428L/N434A/Y436V/Q438K/S440D;    L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and    L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E,    according to EU numbering;-   [38] the Fc region variant of [37], wherein the FcRn-binding domain    comprises a combination of amino acid substitutions selected from    the group consisting of: N434A/Q438R/S440E; N434A/Y436T/Q438R/S440E;    N434A/Y436V/Q438R/S440E; M428L/N434A/Q438R/S440E;    M428L/N434A/Y436T/Q438R/S440E; M428L/N434A/Y436V/Q438R/S440E;    L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and    L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E,    according to EU numbering;-   [39] the Fc region variant of any one of [33] to [38], wherein its    FcRn-binding activity under an acidic pH condition (e.g., pH 5.8) is    enhanced as compared to that of an Fc region of a native IgG;-   [40] the Fc region variant of any one of [33] to [39], wherein its    binding activity to an anti-drug antibody (ADA) is not significantly    enhanced under a neutral pH condition as compared to that of an Fc    region of a native IgG;-   [41] the Fc region variant of [40], wherein the anti-drug antibody    (ADA) is a rheumatoid factor (RF);-   [42] the Fc region variant of any one of [33] to [41], wherein its    plasma clearance (CL) is decreased, plasma retention time is    increased, or plasma half-life (t1/2) is increased, as compared to    that of an Fc region of a native IgG;-   [43] the Fc region variant of any one of [33] to [42], wherein its    plasma retention is increased as compared to a reference Fc region    variant comprising a combination of amino acid substitutions    N434Y/Y436V/Q438R/S440E, according to EU numbering;-   [44] an antibody comprising the Fc region variant of any one of [33]    to [43];-   [45] the antibody of [44], wherein the antibody is an IgG antibody;-   [46] a pharmaceutical composition comprising the antibody of [44] or    [45];-   [47] the pharmaceutical composition of [46], which is for increasing    retention of the antibody in plasma;-   [48] a nucleic acid encoding the Fc region variant of any one of    [33] to [43] or the antibody of [44] or [45];-   [49] a vector comprising the nucleic acid of [48];-   [50] a host cell comprising the vector of [49];-   [51] a method for producing an Fc region variant comprising an    FcRn-binding domain or an antibody comprising the variant, which    comprises culturing the host cell of [50], and then collecting the    Fc region variant or the antibody comprising the variant from the    cell culture;-   [52] the method of [51], which further optionally comprises any one    or more steps selected from the group consisting of:    -   (a) selecting an Fc region variant with enhanced FcRn-binding        activity under an acidic pH condition as compared to that of an        Fc region of a native IgG;    -   (b) selecting an Fc region variant whose binding activity to an        anti-drug antibody (ADA) is not significantly enhanced under a        neutral pH condition as compared to that of an Fc region of a        native IgG;    -   (c) selecting an Fc region variant with increased plasma        retention as compared to that of an Fc region of a native IgG;        and    -   (d) selecting an antibody comprising an Fc region variant that        can promote elimination of an antigen from plasma as compared to        a reference antibody comprising an Fc region of a native IgG;        and-   [53] a method for producing an Fc region variant comprising an    FcRn-binding domain or an antibody comprising the variant, wherein    the method comprises substituting amino acids in a way such that the    resulting Fc region variant or the antibody comprising the variant    comprises Ala at position 434; Glu, Arg, Ser, or Lys at position    438; and Glu, Asp, or Gln at position 440, according to EU    numbering.

In one embodiment, Disclosure B relates to, for example, withoutlimitation:

-   [B1] use of the Fc region variant of any one of [33] to [43] or the    antibody of [44] or [45] in the manufacture of a medicament for    increasing retention in plasma;-   [B2] use of the Fc region variant of any one of [33] to [43] or the    antibody of [44] or [45] in the manufacture of a medicament for not    significantly increasing the binding activity for an anti-drug    antibody (ADA) under a neutral pH condition compared to the Fc    region of a native IgG;-   [B3] use of the Fc region variant of any one of [33] to [43] or the    antibody of [44] or [45] for increasing retention in plasma;-   [B4] use of the Fc region variant of any one of [33] to [43] or the    antibody of [44] or [45] for not significantly increasing the    binding activity for an anti-drug antibody (ADA) under a neutral pH    condition compared to the Fc region of a native IgG; and-   [B5] an Fc region variant or an antibody comprising the variant,    which is obtained by the method of any one of [51], [52], and [53].

According to various embodiments, Disclosure B encompasses combinationsof one or multiple elements described in any of [33] to [53] and [B1] to[B5] mentioned above, in part or as a whole, as long as such acombination is not technically inconsistent with the common technicalknowledge in the art. For example, in some embodiments, Disclosure Bencompasses an Fc region variant comprising an FcRn-binding domain,wherein the FcRn-binding domain can comprise:

(a) Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu,Asp, or Gln at position 440, according to EU numbering;

(b) Ala at position 434; Arg or Lys at position 438; and Glu or Asp atposition 440, according to EU numbering;

(c) Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr,or Phe at position 436; Glu, Arg, Ser, or Lys at position 438; and Glu,Asp, or Gln at position 440, according to EU numbering;

(d) Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr,or Phe at position 436; Arg or Lys at position 438; and Glu or Asp atposition 440, according to EU numbering;

(e) Leu at position 428; Ala at position 434; Val or Thr at position436; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln atposition 440, according to EU numbering; or

(f) Leu at position 428; Ala at position 434; Val or Thr at position436; Arg or Lys at position 438; and Glu or Asp at position 440,according to EU numbering.

In one embodiment, Disclosure C relates to, for example, withoutlimitation:

-   [54] an isolated anti-IL-8 antibody that binds to human IL-8, which    comprises at least one amino acid substitution(s) in at least one    of (a) to (f) below, and binds to IL-8 in a pH-dependent manner:    -   (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:67;    -   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:68;    -   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:69;    -   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:70;    -   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:71;        and    -   (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:72;-   [55] the anti-IL-8 antibody of [54], which comprises an amino acid    substitutions of tyrosine at position 9 of the amino acid sequence    of SEQ ID NO:68, arginine at position 11 of the amino acid sequence    of SEQ ID NO:68, and tyrosine at position 3 of the amino acid    sequence of SEQ ID NO:69;-   [56] the anti-IL-8 antibody of [54] or [55], which further comprises    an amino acid substitutions of alanine at position 6 of the amino    acid sequence of SEQ ID NO:68 and glycine at position 8 of the amino    acid sequence of SEQ ID NO:68;-   [57] the anti-IL-8 antibody of any one of [54] to [56], which    comprises an amino acid substitutions of asparagine at position 1 of    the amino acid sequence of SEQ ID NO:71, leucine at position 5 of    the amino acid sequence of SEQ ID NO:71, and glutamine at position 1    of the amino acid sequence of SEQ ID NO:72;-   [58] the anti-IL-8 antibody of any one of [54] to [57], which    comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID    NO:67, (b) HVR-H2 comprising the amino acid sequence of SEQ ID    NO:73, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID    NO:74;-   [59] the anti-IL-8 antibody of any one of [54] to [58], which    comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID    NO:70, (b) HVR-L2 comprising the amino acid sequence of SEQ ID    NO:75, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID    NO:76;-   [60] the anti-IL-8 antibody of any one of [54] to [59], which    comprises the heavy chain variable region of SEQ ID NO:78 and the    light chain variable region of SEQ ID NO:79;-   [61] the anti-IL-8 antibody of any one of [54] to [60], which    comprises an Fc region having at least one property selected from    the properties of (a) to (f) below:    -   (a) increased binding affinity for FcRn of the Fc region        relative to the binding affinity for FcRn of a native Fc region        at acidic pH;    -   (b) reduced binding affinity of the Fc region for pre-existing        ADA relative to the binding affinity of a native Fc region for        the pre-existing ADA;    -   (c) increased plasma half-life of the Fc region relative to the        plasma half-life of a native Fc region;    -   (d) reduced plasma clearance of the Fc region relative to the        plasma clearance of a native Fc region; and    -   (e) reduced binding affinity of the Fc region for an effector        receptor relative to the binding affinity of a native Fc region        for the effector receptor; and    -   (f) increased binding to extracellular matrix.-   [62] the anti-IL-8 antibody of [61], wherein the Fc region comprises    amino acid substitution(s) at one or more positions selected from    the group consisting of position 235, 236, 239, 327, 330, 331, 428,    434, 436, 438 and 440, according to EU numbering;-   [63] the anti-IL-8 antibody of [62], which comprises an Fc region    comprising one or more amino acid substitutions selected from the    group consisting of L235R, G236R, S239K, A327G, A330S, P331S, M428L,    N434A, Y436T, Q438R and S440E;-   [64] the anti-IL-8 antibody of [63], wherein the Fc region comprises    the amino acid substitutions of L235R, G236R, S239K, M428L, N434A,    Y436T, Q438R and S440E;-   [65] the anti-IL-8 antibody of [63], wherein the Fc region comprises    the amino acid substitution of L235R, G236R, A327G, A330S, P331S,    M428L, N434A, Y436T, Q438R and S440E;-   [66] an anti-IL-8 antibody that comprises a heavy chain comprising    the amino acid sequence of SEQ ID NO:81 and a light chain comprising    the amino acid sequence of SEQ ID NO:82;-   [67] an anti-IL-8 antibody that comprises a heavy chain comprising    the amino acid sequence of SEQ ID NO:80 and a light chain comprising    the amino acid sequence of SEQ ID NO:82;-   [68] an isolated nucleic acid encoding the anti-IL-8 antibody of any    one of [54] to [67];-   [69] a vector comprising the nucleic acid of [68];-   [70] a host cell comprising the vector of [69];-   [71] a method for producing an anti-IL-8 antibody, which comprises    culturing the host of [70];-   [72] the method for producing an anti-IL-8 antibody of [71], which    comprises isolating the antibody from a culture supernatant;-   [73] a pharmaceutical composition comprising the anti-IL-8 antibody    of any one of-   [54] to [67], and a pharmaceutically acceptable carrier;-   [74] the anti-IL-8 antibody of any one of [54] to [67] for use in a    pharmaceutical composition;-   [75] the anti-IL-8 antibody of any one of [54] to [67] for use in    the treatment of a disorder with the presence of excess IL-8;-   [76] use of the anti-IL-8 antibody of any one of [54] to [67] in the    manufacture of a pharmaceutical composition for a disorder with the    presence of excess IL-8;-   [77] a method for treating a patient that has a disorder with the    presence of excess IL-8, which comprises administering the anti-IL-8    antibody of any one of [54] to [67] to the individual;-   [78] a method for promoting elimination of IL-8 from an individual,    which comprises administering the anti-IL-8 antibody of any one of    [54] to [67] to the individual;-   [79] a pharmaceutical composition comprising the anti-IL-8 antibody    of any one of [54] to [67], wherein the antibody binds to IL-8 and    binds to extracellular matrix; and-   [80] a method for producing an anti-IL-8 antibody comprising a    variable region with a pH-dependent IL-8-binding activity, wherein    the method comprises:    -   (a) evaluating binding of an anti-IL-8 antibody with        extracellular matrix,    -   (b) selecting an anti-IL-8 antibody with strong binding to the        extracellular matrix,    -   (c) culturing a host that comprises a vector comprising a        nucleic acid encoding the antibody, and    -   (d) isolating the antibody from the culture solution.

In an alternative embodiment, Disclosure C relates to:

-   [C1] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] in the manufacture of a pharmaceutical composition    for suppressing accumulation of IL-8 which has a biological    activity;-   [C2] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] for suppressing accumulation of IL-8 which has a    biological activity;-   [C3] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] in the manufacture of a pharmaceutical composition    for inhibiting angiogenesis;-   [C4] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] for inhibiting angiogenesis;-   [C5] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] in the manufacture of a pharmaceutical composition    for inhibiting facilitation of neutrophil migration;-   [C6] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] for inhibiting facilitation of neutrophil migration;-   [C7] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to    [C31] for use in suppressing accumulation of IL-8 which has a    biological activity;-   [C8] a method for suppressing accumulation of IL-8 which has a    biological activity, wherein the method comprises administering the    anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] to    an individual;-   [C9] a pharmaceutical composition for suppressing accumulation of    IL-8 which has a biological activity, comprising the anti-IL-8    antibody of any one of [54] to [67] and [C26] to [C31];-   [C10] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to    [C31] for use in inhibiting angiogenesis;-   [C11] a method for inhibiting angiogenesis in an individual, wherein    the method comprises administering the anti-IL-8 antibody of any one    of [54] to [67] and [C26] to [C31] to the individual;-   [C12] a pharmaceutical composition for inhibiting angiogenesis,    which comprises the anti-IL-8 antibody of any one of [54] to [67]    and [C26] to [C31];-   [C13] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to    [C31] for use in inhibiting facilitation of neutrophil migration;-   [C14] a method for inhibiting facilitation of neutrophil migration    in an individual, wherein the method comprises administering the    anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] to    the individual;-   [C15] a pharmaceutical composition for inhibiting facilitation of    neutrophil migration, which comprises the anti-IL-8 antibody of any    one of [54] to [67] and [C26] to [C31];-   [C16] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to    [C31] for use in the treatment of a disorder with the presence of    excess IL-8;-   [C17] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] in the manufacture of a pharmaceutical composition    for treating a disorder with the presence of excess IL-8;-   [C18] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] for treating a disorder with the presence of excess    IL-8;-   [C19] a method for treating a disorder with the presence of excess    IL-8 in an individual, wherein the method comprises administering    the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31]    to the individual;-   [C20] a pharmaceutical composition for treating a disorder with the    presence of excess IL-8, which comprises the anti-IL-8 antibody of    any one of [54] to [67] and [C26] to [C31];-   [C21] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to    [C31] for use in promoting elimination of IL-8;-   [C22] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] in the manufacture of a pharmaceutical composition    for promoting elimination of IL-8;-   [C23] use of the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] for promoting elimination of IL-8;-   [C24] a method for promoting elimination of IL-8 in an individual,    wherein the method comprises administering the anti-IL-8 antibody of    any one of [54] to [67] and [C26] to [C31] to the individual; and-   [C25] a pharmaceutical composition for promoting elimination of    IL-8, which comprises the anti-IL-8 antibody of any one of [54] to    [67] and [C26] to [C31].-   [C26] An anti-IL-8 antibody, which comprises an Fc region comprising    amino acid substitution(s) at one or more positions selected from    the group consisting of position 235, 236, 239, 327, 330, 331, 428,    434, 436, 438 and 440, according to EU numbering.-   [C27] The anti-IL-8 antibody of [C26], which comprises an Fc region    having at least one property selected from the properties of (a)    to (f) below:    -   (a) increased binding affinity for FcRn of the Fc region        relative to the binding affinity for FcRn of a native Fc region        at acidic pH;    -   (b) reduced binding affinity of the Fc region for pre-existing        ADA relative to the binding affinity of a native Fc region for        the pre-existing ADA;    -   (c) increased plasma half-life of the Fc region relative to the        plasma half-life of a native Fc region;    -   (d) reduced plasma clearance of the Fc region relative to the        plasma clearance of a native Fc region;    -   (e) reduced binding affinity of the Fc region for an effector        receptor relative to the binding affinity of a native Fc region        for the effector receptor; and    -   (f) increased binding to extracellular matrix.-   [C28] The anti-IL-8 antibody of [C26] or [C27], which comprises an    Fc region comprising one or more amino acid substitution(s) selected    from the group consisting of L235R, G236R, S239K, A327G, A330S,    P331S, M428L, N434A, Y436T, Q438R and S440E, according to EU    numbering.-   [C29] The anti-IL-8 antibody of [C28], which comprises an Fc region    comprising one or more amino acid substitutions selected from the    group consisting of (a) L235R, G236R, S239K, M428L, N434A, Y436T,    Q438R and S440E; or (b) L235R, G236R, A327G, A330S, P331S, M428L,    N434A, Y436T, Q438R and S440E, according to EU numbering.-   [C30] The anti-IL-8 antibody of [C26] that comprises a heavy chain    comprising the amino acid sequence of SEQ ID NO:81 and a light chain    comprising the amino acid sequence of SEQ ID NO:82.-   [C31] The anti-IL-8 antibody of [C26] that comprises a heavy chain    comprising the amino acid sequence of SEQ ID NO:80 and a light chain    comprising the amino acid sequence of SEQ ID NO:82.-   [C32] An isolated nucleic acid encoding the anti-IL-8 antibody of    any one of [C26] to [C31].-   [C33] A vector comprising the nucleic acid of [C32].-   [C34] A host cell comprising the vector of [C33].-   [C35] A method for producing an anti-IL-8 antibody, which comprises    culturing the host cell of [C34].-   [C36] The method for producing an anti-IL-8 antibody of any one of    [C26] to [C31], which further comprises isolating the antibody from    the host cell culture.-   [C37] A pharmaceutical composition comprising the anti-IL-8 antibody    of any one of [C26] to [C31] and a pharmaceutically acceptable    carrier.-   [C38] A method for treating a patient that has a disorder with the    presence of excess IL-8, which comprises administering the anti-IL-8    antibody of any one of [C26] to [C31] to the individual.-   [C39] A method for promoting elimination of IL-8 from an individual,    which comprises administering the anti-IL-8 antibody of any one of    [C26] to [C31] to the individual.-   [C40] A method for inhibiting IL-8, wherein the method comprises    contacting the anti-IL-8 antibody of any one of [54] to [67] and    [C26] to [C31] with IL-8.-   [C41] The method of [C40], wherein the method inhibits a biological    activity of IL-8.

According to various embodiments, Disclosure C encompasses combinationsof one or multiple elements described in any of [54] to [80] and [C1] to[C41] mentioned above, in part or as a whole, as long as such acombination is not technically inconsistent with the common technicalknowledge in the art.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows changes in the plasma concentration of human IL-6 receptorin human FcRn transgenic mice administered with a human IL-6receptor-binding antibody that binds to human IL-6 receptor in apH-dependent manner and whose constant region is that of a native IgG1(Low_pI-IgG1), or an antibody that has increased the pI of the variableregion in the antibody (High_pI-IgG1).

FIG. 2 shows changes in the plasma concentration of human IL-6 receptorin human FcRn transgenic mice administered individually with a humanIL-6 receptor-binding antibody that binds to human IL-6 receptor in apH-dependent manner and has been conferred with binding to FcRn under aneutral pH condition (Low_pI-F939), and antibodies that have increasedthe pI of the variable region in the antibody (Middle_pI-F939,High_pI-F939).

FIG. 3 shows changes in the plasma concentration of human IL-6 receptorin human FcRn transgenic mice administered individually with a humanIL-6 receptor-binding antibody that binds to human IL-6 receptor in apH-dependent manner and whose FcγR binding under a neutral pH conditionis increased (Low_pI-F1180), and antibodies that have increased the pIof the variable region in the antibody (Middle_pI-F1180, High_pI-F1180).

FIG. 4 shows changes in the plasma concentration of human IL-6 receptorin human FcRn transgenic mice whose soluble human IL-6 receptorconcentration in plasma is maintained at a steady state, which have beenadministered individually with a human IL-6 receptor-binding antibodythat binds to human IL-6 receptor in a pH-dependent manner and whoseconstant region is that of a native IgG1 (Low_pI-IgG1), an antibody thatcomprises an Fc region variant in which the Fc region in the antibodyhas increased FcRn binding under a neutral pH condition (Low_pI-F11),and antibodies that have increased the pI of the variable region inthese antibodies (High_pI-IgG1, High_pI-F11).

FIG. 5 shows the extent of extracellular matrix binding of each of thethree types of antibodies with different pIs that bind to human IL-6receptor in a pH-dependent manner (Low_pI-IgG1, Middle_pI-IgG1 andHigh_pI-IgG1) and the two types of antibodies with different pIs that donot bind to human IL-6 receptor in a pH-dependent manner(Low_pI(NPH)-IgG1 and High_pI(NPH)-IgG1). “NPH” means pH independentwithin the scope of Disclosure A described herein.

FIG. 6 shows relative values of the extent of soluble human FcγRIIbbinding (measured by BIACORE®) of antibodies that comprise an Fc regionvariant each of whose pI has been increased by modifying one amino acidresidue in the constant region of the Ab1H-P600 antibody which binds toIgE in a pH-dependent manner, by setting the value of Ab1H-P600 to 1.00.

FIG. 7 shows relative values of the rate at which antibodies thatcomprise an Fc region variant each of whose pI has been increased bymodifying one amino acid residue in the constant region of Ab1H-P600 aretaken up into cells of an hFcγRIIb-expressing cell line, respectively,evaluated with the value of Ab1H-P600 set to 1.00.

FIG. 8 shows the extent of binding of Fv4-IgG1, which has the Fc regionof a native human IgG1, to rheumatoid factor in the serum of each RApatient.

FIG. 9 shows the extent of binding of Fv4-YTE, which has an Fc regionvariant with increased FcRn binding, to rheumatoid factor in the serumof each RA patient.

FIG. 10 shows the extent of binding of Fv4-LS, which has an Fc regionvariant with increased FcRn binding, to rheumatoid factor in the serumof each RA patient.

FIG. 11 shows the extent of binding of Fv4-N434H, which has an Fc regionvariant with increased FcRn binding, to rheumatoid factor in the serumof each RA patient.

FIG. 12 shows the extent of binding of Fv4-F1847m, which has an Fcregion variant with increased FcRn binding, to rheumatoid factor in theserum of each RA patient.

FIG. 13 shows the extent of binding of Fv4-F1848m, which has an Fcregion variant with increased FcRn binding, to rheumatoid factor in theserum of each RA patient.

FIG. 14 shows the extent of binding of Fv4-F1886m, which has an Fcregion variant with increased FcRn binding, to rheumatoid factor in theserum of each RA patient.

FIG. 15 shows the extent of binding of Fv4-F1889m, which has an Fcregion variant with increased FcRn binding, to rheumatoid factor in theserum of each RA patient.

FIG. 16 shows the extent of binding of Fv4-F1927m, which has an Fcregion variant with increased FcRn binding, to rheumatoid factor in theserum of each RA patient.

FIG. 17 shows the extent of binding of Fv4-F1168m, which has an Fcregion variant with increased FcRn binding, to rheumatoid factor in theserum of each RA patient.

FIG. 18 shows average values of the binding of Fv4-IgG1, which has theFc region of a native human IgG1, and each of the antibodies comprisinga novel Fc region variant in which the Fc region has an Fc regionvariant with increased binding to each FcRn, to rheumatoid factor in theserum of RA patients.

FIG. 19 shows changes in the plasma concentration of each anti-human IgEantibody in cynomolgus when administered with OHB-IgG1 which is ananti-human IgE antibody and has the Fc region of a native human IgG1,and each of the antibodies comprising a novel Fc region variant in whicheach the Fc region has an Fc region variant with increased binding toFcRn (OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889mand OHB-F1927m).

FIG. 20 shows changes in the plasma concentration of an anti-human IL-6receptor antibody in human FcRn transgenic mouse when administered withFv4-IgG1 which is an anti-human IL-6 receptor antibody and has the Fcregion of a native human IgG1, or Fv4-F1718 which has increased FcRnbinding of the antibody at the acidic pH condition.

FIG. 21 shows sensorgrams obtained for IL-8 binding of H998/L63 and Hr9at pH 7.4 and pH 5.8 measured with Biacore.

FIG. 22 shows changes of the human IL-8 concentration in mouse plasmawhen H998/L63 or H89/L118 was administered to mice at 2 mg/kg in amixture with human IL-8.

FIG. 23 shows changes of the human IL-8 concentration in mouse plasmawhen H89/L118 was administered to mice at 2 mg/kg or 8 mg/kg in amixture with human IL-8.

FIG. 24 shows changes of the human IL-8 concentration in mouse plasmawhen H89/L118 or H553/L118 was administered to mice at 2 mg/kg or 8mg/kg in a mixture with human IL-8.

FIG. 25A shows changes in the relative values of antibodyconcentration-dependent chemiluminescence with antibody Hr9, H89/L118 orH553/L118 before preservation in plasma.

FIG. 25B shows changes in the relative values of antibodyconcentration-dependent chemiluminescence with antibody Hr9, H89/L118 orH553/L118 after one week of preservation in plasma.

FIG. 25C shows changes in the relative values of antibodyconcentration-dependent chemiluminescence with antibody Hr9, H89/L118 orH553/L118 after two weeks of preservation in plasma.

FIG. 26 shows the predicted frequency of ADA occurrence for eachanti-IL-8 antibody (hWS4, Hr9, H89/L118, H496/L118 or H553/L118) and thepredicted frequency of ADA occurrence for other pre-existing therapeuticantibodies predicted by the EpiMatrix.

FIG. 27 shows the predicted frequency of ADA occurrence for eachanti-IL-8 antibody (H496/L118, H496v1/L118, H496v2/L118, H496v3/L118,H1004/L118 or H1004/L395) and the predicted frequency of ADA occurrencefor other pre-existing therapeutic antibodies predicted by EpiMatrix.

FIG. 28A shows changes in the relative values of antibodyconcentration-dependent chemiluminescence with antibody Hr9, H89/L118 orH1009/L395-F1886s before preservation in plasma.

FIG. 28B shows changes in the relative values of antibodyconcentration-dependent chemiluminescence with antibody Hr9, H89/L118 orH1009/L395-F1886s after one week of preservation in plasma.

FIG. 28C shows changes in the relative values of antibodyconcentration-dependent chemiluminescence with antibody Hr9, H89/L118 orH1009/L395-F1886s after two weeks of preservation in plasma.

FIG. 29 shows changes of the human IL-8 concentration in mouse plasmawhen mice were administered with each of H1009/L395, H553/L118 andH998/L63 in a mixture with human IL-8.

FIG. 30 shows the extent of extracellular matrix binding when Hr9,H89/L118 or H1009/L395 was added alone to extracellular matrix, and whenthey were added in a mixture with human IL-8.

FIG. 31 shows changes of antibody concentration in mouse plasma when anantibody that has the variable region of H1009/L395 and the Fc regionthat does not bind to FcRn (F1942m) was administered alone or in amixture with human IL-8 to human FcRn transgenic mice.

FIG. 32 shows the predicted frequency of ADA occurrence for H1009/L395and H1004/L395 and the predicted frequency of ADA occurrence for otherpre-existing therapeutic antibodies predicted by EpiMatrix.

FIG. 33 shows changes in the concentration of the respective anti-humanIL-8 antibody in the plasma of cynomolgus when administered withH89/L118-IgG1, which has the variable region of H89/L118 and the Fcregion of a native human IgG1, and each antibody that has an Fc regionvariant with increased binding to FcRn (H89/L118-F1168m,H89/L118-F1847m, H89/L118-F1848m, H89/L118-F1886m, H89/L118-F1889m andH89/L118-F1927m).

FIG. 34 shows the binding of antibodies that have the variable region ofH1009/L395 and whose Fc region is a variant (F1886m, F1886s, or F1974m)to each FcγR.

FIG. 35 shows changes of the human IL-8 concentration in mouse plasmawhen an anti-IL-8 antibody was administered to human FcRn transgenicmice in a mixture with human IL-8. In this case, the anti-IL-8 antibodywas H1009/L395-IgG1 (2 mg/kg) which comprises the variable region ofH1009/L395 and the Fc region of a native human IgG1, orH1009/L395-F1886s (2, 5 or 10 mg/kg) which comprises the variable regionof H1009/L395 and the modified Fc region.

FIG. 36 shows changes in the antibody concentration in the plasma ofcynomolgus when administered with Hr9-IgG1 or H89/L118-IgG1, both ofwhich comprise the Fc region of a native human IgG1, orH1009/L395-F1886s or H1009/L395-F1974m, both of which comprise amodified Fc region.

FIG. 37 shows the IgE plasma concentration time profile of some anti-IgEantibodies in C57BL6J mice in terms of the antibody variable regionmodification.

FIG. 38 (FIGS. 38A-38D) shows Octet sensorgrams of selected 25 [twentyfive] pH-dependent and/or calcium-dependent antigen binding clones.

FIG. 39 shows the C5 plasma concentration time profile of some anti-C5bispecific antibodies in C57BL6J mice in terms of the antibody variableregion modification.

FIG. 40 shows the IgE plasma concentration time profile of some anti-IgEantibodies in C57BL6J mice in terms of the antibody constant regionmodification.

DETAILED DESCRIPTION

Non-limiting embodiments of Disclosure A, B or C are describedhereinbelow. All embodiments described in the Examples hereinbelow aredescribed with the intention to be rightfully understood to be alsodescribed in the section on “DETAILED DESCRIPTION”, without constraintsby any patent practices, ordinance, regulations, or others that may beattempted to narrowly interpret the contents described in the Examplesin countries where acquisition of patent right from the present patentapplication is intended.

Disclosure A or Disclosure B

In some embodiments, Disclosure A relates to antibodies comprising anantigen-binding domain whose antigen-binding activity changes accordingto ion concentration conditions, in which the isoelectric point (pI) isincreased by modification of at least one amino acid residue that may beexposed on the antibody surface (herein, also referred to as “ionconcentration-dependent antibodies with increased pI” within the scopeof Disclosure A; and the antigen-binding domains of the antibodies arealso referred to as “ion concentration-dependent antigen-binding domainswith increased pI”). The invention is partly based on the surprisingdiscovery of the inventors that antigen elimination from plasma can befacilitated with an ion concentration-dependent antibody whoseisoelectric point (pI) has been increased by the modification of atleast one amino acid residue that can be exposed on the antibody surface(for example, when the antibody is administered in vivo); and thatbinding of an antibody to the extracellular matrix can be increased withan ion concentration-dependent antibody with increased (elevated) pI.The invention is also partly based on the surprising discovery of theinventors that this beneficial effect is brought about by combining twoentirely different concepts of: an ion concentration-dependentantigen-binding domain or ion concentration-dependent antibody; and anantibody whose pI is increased by modification of at least one aminoacid residue that can be exposed on the surface (herein, also referredto as an “antibody with increased pI” within the scope of Disclosure A;and an antibody whose pI is decreased (reduced) by modification of atleast one amino acid residue that can be exposed on the surface isreferred to as an “antibody with decreased pI” within the scope ofDisclosure A). The invention is thus categorized as a type of pioneerinvention which can lead to remarkable technological innovation in thefield (e.g., medical field) to which Disclosure A belongs.

As a matter of course, for example, an antibody comprising anantigen-binding domain and whose pI is increased by modification of atleast one amino acid residue that can be exposed on the antibodysurface, which has been further modified so that the antigen-bindingactivity of the antigen-binding domain changes according to ionconcentration conditions, are also included within the scope ofDisclosure A described herein (herein, such antibody is also referred toas an “ion concentration-dependent antibody with increased pI” withinthe scope of the Disclosure A).

As a matter of course, for example, an antibody containing an ionconcentration-dependent antigen-binding domain in which at least oneamino acid residue that can be exposed on the antibody surface has acharge different from that of the at least one amino acid residue at thecorresponding position(s) in an antibody before modification (nativeantibody (for example, native Ig antibody, preferably native IgGantibody), or reference or parent antibody (e.g., antibody beforemodification, or antibody prior to or during library construction, orthe like)), and whose net antibody pI is increased is also included inDisclosure A described herein (such antibody is also referred to as an“ion concentration-dependent antibody with increased pI” within thescope of Disclosure A described herein).

As a matter of course, for example, an antibody containing an ionconcentration-dependent antigen-binding domain, whose pI is increased bymodification of at least one amino acid residue that can be exposed onthe antibody surface in an antibody before the modification (nativeantibody (for example, native Ig antibody, preferably native IgGantibody, or reference or parent antibody (e.g., antibody before themodification, or antibody prior to or during library construction, orthe like)) is also included in Disclosure A described herein (suchantibody is also referred to as an “ion concentration-dependent antibodywith increased pI” within the scope of Disclosure A described herein).

As a matter of course, for example, an antibody containing an ionconcentration-dependent antigen-binding domain in which at least oneamino acid residue that can be exposed on the antibody surface ismodified for the purpose of increasing the pI of the antibody is alsoincluded in Disclosure A described herein (such antibody is alsoreferred to as an “ion concentration-dependent antibody with increasedpI” within the scope of Disclosure A described herein).

Within the scope of Disclosures A and B described herein, “amino acids”include not only natural amino acids but also unnatural amino acids.Within the scope of Disclosures A and B described herein, amino acids oramino acid residues may be represented by either one-letter (forexample, A) or three-letter codes (for example, Ala), or both (forexample, Ala(A)).

As used in the context of Disclosures A and B, “modification of an aminoacid”, “modification of an amino acid residue”, or an equivalent phrasemay be understood as, without being limited thereto, chemicallymodifying one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10) specific amino acids (residues) in an antibody amino acidsequence with a molecule or adding, deleting, substituting or insertingone or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10) amino acids in an antibody amino acid sequence. Amino acid addition,deletion, substitution, or insertion can be carried out to a nucleicacid encoding an amino acid sequence, for example, by site-directedmutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492(1985)) or overlap extension PCR; via affinity maturation of antibodies,or by using chain shuffling of antibody heavy chains or light chains; orby antigen panning-based selection using phage-display libraries (Smithet al., Methods Enzymol. 217:228-257 (1993)); and these can be performedalone or in appropriate combinations. Such amino acid modification iscarried out preferably, without limitation, by substituting one or moreamino acid residue in an antibody amino acid sequence with a differentamino acid (individually). Amino acid addition, deletion, substitution,or insertion, and modification of an amino acid sequence by humanizationor chimerization can be carried out by methods known in the art.Alteration or modification of an amino acid (residue), such as aminoacid addition, deletion, substation, or insertion, may also be performedon an antibody variable region or an antibody constant region to be usedin preparing recombinant antibodies for the antibodies of Disclosure Aor B.

In one embodiment within the scope of Disclosures A and B describedherein, substitution of amino acids (residues) refers to substitutionwith different amino acids (residues), and can be designed to modify,for example, matters such as in (a) to (c): (a) the polypeptide backbonestructure in a region of sheet or helical conformation; (b) charge orhydrophobicity at a target site; or (c) size of a side chain.

Amino acid residues are classified, based on properties of the sidechains in the structure, for example, into the groups of: (1)hydrophobic: norleucine, Met, Ala, Val, Leu, and Ile; (2) neutral,hydrophilic: Cys, Ser, Thr, Asn, and Gln; (3) acidic: Asp and Glu; (4)basic: His, Lys, and Arg; (5) residues that affect the chainorientation: Gly and Pro; and (6) aromatic: Trp, Tyr, and Phe.

Substitution of amino acid residues within each group is referred to asconservative substitution, while substitution of amino acid residuesbetween different groups is referred to as non-conservativesubstitution. Substitution of amino acid residues may be conservativesubstitution, non-conservative substitution, or a combination thereof.Several known appropriate methods may be used for substituting aminoacids with those other than natural amino acids (Wang et al., Annu. Rev.Biophys. Biomol. Struct. 35:225-249 (2006); Forster et al., Proc. Natl.Acad. Sci. USA 100(11):6353-6357 (2003)). It is possible to use, forexample, a cell-free translation system containing tRNA in which anunnatural amino acid is linked to amber suppressor tRNA complementary toUAG codon (amber codon) which is a stop codon (Clover Direct (ProteinExpress)).

Within the scope of Disclosures A and B described herein, it isunderstood that the structure of an “antigen” is not limited to aspecific structure as long as the antigen includes an epitope that bindsto an antibody. The antigen may be an inorganic or organic substance.Antigens may be any ligands, including various cytokines, for example,interleukins, chemokines, and cell growth factors. Alternatively, as amatter of course, for example, receptors that are present as in asoluble form or have been modified to be a soluble form in biologicalfluids such as plasma can also be used as antigens. Non-limitingexamples of such soluble receptors include the soluble IL-6 receptordescribed in Müllberg et al., J. Immunol. 152(10):4958-4968 (1994).Furthermore, antigens may be monovalent (for example, soluble IL-6receptor) or multivalent (for example, IgE).

In one embodiment, antigens that can be bound by an antibody ofDisclosures A and B are preferably soluble antigens present inbiological fluids (for example, biological fluids illustrated inWO2013/125667, preferably plasma, interstitial fluid, lymphatic fluid,ascitic fluid, or pleural fluid) of subjects (within the scope ofDisclosures A and B described herein, subjects to be administered(applied) with the antibody, which can be virtually any animal, forexample, a human, mouse, etc.); however, the antigens may also bemembrane antigens.

Within the scope of Disclosures A and B described herein, “prolongationof the half-life in plasma” or “shortening of the half-life in plasma”of a target molecule (which may be an antigen or antibody), or anequivalent phrase thereof can also be represented more specificallyusing in addition to the parameter of half-life in plasma (t1/2), anyother parameter such as mean retention time in plasma, clearance (CL) inplasma, and area under the concentration curve (AUC) (Pharmacokinetics:Enshuniyoru Rikai (Understanding through practice) Nanzando). Theseparameters can be specifically assessed, for example, by carrying outnoncompartmental analysis according to the protocol appended to the invivo kinetics analysis software WinNonlin (Pharsight). It is known tothose of ordinary skill in the art that these parameters normallycorrelate with one another.

Within the scope of Disclosures A and B described herein, an “epitope”refers to an antigenic determinant in an antigen and means a site on anantigen at which the antigen-binding domain of an antibody binds. Thus,an epitope can be defined, for example, based on its structure.Alternatively, the epitope may be defined by the antigen-bindingactivity of an antibody that recognizes the epitope. When an antigen isa peptide or polypeptide, the epitope can be specified by the amino acidresidues that constitute the epitope. Alternatively, when an epitope isa sugar chain, the epitope can be specified based on its specific sugarchain structure. An antigen-binding domain of Disclosures A and B maybind to a single epitope or different epitopes on an antigen.

A linear epitope may be a primary amino acid sequence. Such a linearepitope typically contains at least three and commonly at least five,for example, 8 to 10 amino acids or 6 to 20 amino acids as a uniquesequence.

In a conformational epitope, typically the amino acids that constitutethe epitope are not present consecutively as a primary sequence. Anantibody can recognize a conformational epitope in the three-dimensionalstructure of a peptide or protein. Methods for determining theconformation of an epitope include, but are not limited to, X raycrystallography, two-dimensional nuclear magnetic resonance,site-specific spin labeling, and electron paramagnetic resonance(Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol.66, Morris (ed.)).

Within the scope of Disclosures A and B described herein, an “antibody”is not particularly limited and used in the broadest sense, as long asit can bind to an antigen of target. Non-limiting examples of antibodiesinclude widely known common antibodies (for example, nativeimmunoglobulins (abbreviated as “Ig”)), and molecules and variantsderived therefrom, for example, Fab, Fab′, F(ab′)₂, diabodies, ScFv(Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993);EP404,097; WO93/11161; Peer et al., Nature Nanotechnology 2:751-760(2007)), low molecular weight antibodies (minibodies) (Orita et al.,Blood 105:562-566 (2005)), scaffold proteins, one-armed antibodies(including all embodiments of one-armed antibodies described inWO2005/063816), multispecific antibodies (for example, bispecificantibodies: antibodies with specificity to two different epitopes,including antibodies that recognize different antigens and antibodiesthat recognize different epitopes on a same antigen). Within the scopeof Disclosures A and B described herein, “bispecific antibodies” are notlimited but may be prepared, for example, as antibody molecules havingthe common L chain described in WO2005/035756, or by the methoddescribed in WO2008/119353 where two general types of antibodies havingan IgG4-like constant regions are mixed to cause an exchange reactionbetween the two types of such antibodies (known as the “Fab-armexchange” method to those of ordinary skill in the art). In analternative embodiment, they may be antibodies having a structure wherethe heavy-chain variable region and the light-chain variable region arelinked together as a single chain (for example, sc(Fv)₂). Alternatively,they may be antibody-like molecules (for example, scFv-Fc) that resultfrom linking the Fc region (a constant region that lacks the CH1 domain)to scFv (or sc(Fv)₂) where the heavy-chain variable region (VH) islinked to the light-chain variable region (VL). Multispecific antibodiesconsisting of scFv-Fc have an (scFv)₂-Fc structure where the first andsecond polypeptides are VH1-linker-VL1-Fc and VH2-linker-VL2-Fc,respectively. Alternatively, they may be antibody-like molecules where asingle-domain antibody is linked to an Fc region (Marvin et al., Curr.Opin. Drug Discov. Devel. 9(2):184-193 (2006)), Fc fusion proteins (forexample, immunoadhesin) (US2013/0171138), functional fragments thereof,substances functionally equivalent thereto, and sugar chain-modifiedvariants thereof. Herein, native IgG (e.g. native IgG1) refers topolypeptides that contain the same amino acid sequence as that ofnaturally occurring IgG (e.g. native IgG1) and belongs to the class ofantibodies encoded substantially by the immunoglobulin gamma gene.Native IgG may be spontaneous mutants thereof and the like.

Typically, where an antibody has a structure that is substantially thesame as or similar to that of native IgG, the Y-shaped structure of thefour chains (two heavy chain polypeptides and two light chainpolypeptides) can be the basic structure. Typically, the heavy chain andthe light chain can be linked together via a disulfide bond (SS bond)and form a heterodimer. Such heterodimers may be linked together via adisulfide bond and form a Y-shaped heterotetramer. The two heavy chainsor light chains may be identical or different from each other.

For example, an IgG antibody may be cleaved into two units of Fab(region) and a single unit of Fc (region) by papain digestion, whichcleaves the hinge region (also referred to as the “hinge” within thescope of Disclosures A and B described herein) where the heavy-chain Fabregion is linked to the Fc region. Typically, the Fab region contains anantigen-binding domain. Since phagocytic cells such as leukocytes andmacrophages have receptors that are capable of binding to the Fc region(Fc receptors), and can recognize via the Fc receptors antibodies thatare bound to an antigen and phagocytize the antigen (opsonization).Meanwhile, the Fc region is involved in the mediation of immunereactions such as ADCC or CDC, and has an effector function of inducinga reaction upon an antibody binding to antigens. The antibody effectorfunction is known to vary according to the type of immunoglobulin(isotype). The Fc region of the IgG class would indicate a region thatspans, for example, from cysteine of position 226 or from proline ofposition 230 (EU numbering) to the C terminus; however, the Fc region isnot limited thereto. The Fc region can be appropriately obtained bypartial digestion of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody, orothers, with a protease such as pepsin, followed by elution of adsorbedfractions from a protein A or protein G column.

Within the scope of Disclosures A and B described herein, the positionsof amino acid residues in the variable region (CDR(s) and/or FR(s)) ofan antibody are shown according to Kabat, whereas the positions of aminoacid residues in the constant region or Fc region are shown according toEU numbering based on Kabat's amino acid positions (Sequences ofProteins of Immunological Interest (National Institute of Health,Bethesda, Md., 1987 and 1991).

Within the scope of Disclosures A and B described herein, “library” mayrefer to molecules (populations) such as multiple antibodies that havesequence variability, in which their respective sequences may be thesame or different from one another; multiple fusion polypeptidescontaining the antibodies; or nucleic acids or polynucleotides encodingthese amino acid sequences, as described in detail in WO2013/125667 (forexample, paragraphs 0121-0125). The library may, for example, contain atleast 10⁴ antibody molecules, more preferably, at least 10⁵ antibodymolecules, even more preferably, at least 10⁶ antibody molecules,particularly preferably, at least 10⁷ antibody molecules or more. Thelibrary may be phage libraries. The phrase “primarily consist of” meansthat antibodies which may have different antigen-binding activitiesaccount for a certain portion among the numerous independent clones withdifferent sequences in the library. In one embodiment, immune librariesthat are constructed based on antibody genes derived from lymphocytes ofanimals immunized with a specific antigen, patients with infection,humans with elevated antibody titer in blood due to vaccination, orpatients with cancer or autoimmune disease can be appropriately used asrandomized variable region libraries. In an alternative embodiment,naive libraries containing naive sequences (antibody sequences withoutbias in the repertoire), which are constructed from antibody genesderived from lymphocytes of healthy persons, can also be appropriatelyused as randomized variable region libraries (Gejima et al., HumanAntibodies 11:121-129 (2002)); Cardoso et al., Scand. J. Immunol.51:337-344 (2000)). Amino acid sequences containing naive sequences canrefer to those obtained from such naive libraries. In an alternativeembodiment, synthetic libraries in which the CDR sequence from a V geneof genomic DNA or a reconstructed functional V gene is substituted witha set of synthetic oligonucleotides containing a sequence encoding acodon set of appropriate length can also be appropriately used asrandomized variable region libraries. In this case, it is also possibleto substitute only the heavy chain CDR3 sequence, since sequencevariations are observed in the CDR3 gene. A standard way to produceamino acid diversity in the antibody variable region may be to increasevariations of amino acid residues at positions that can be exposed onthe antibody surface.

In one embodiment, where antibodies of Disclosure A or B, for example,have a structure that is substantially the same as or similar to thestructure of native Ig antibodies, they typically have variable regions(“V regions”) [heavy chain variable region (“VH region”) and light chainvariable region (“VL region”)] and constant regions (“C regions”)[“heavychain constant region (“CH region”) and light chain constant region (“CLregion”)]. The CH region is further divided into three: CH1 to CH3.Typically, the Fab region of the heavy chain contains VH region and CH1,and typically the Fc region of the heavy chain contains CH2 and CH3.Typically, the hinge region is located between CH1 and CH2. Furthermore,the variable region typically has complementarity determining regions(“CDRs”) and framework regions (“FRs”). Typically, the VH region and VLregion each have three CDRs (CDR1, CDR2, and CDR3) and four FRs (FR1,FR2, FR3, and FR4). Typically, the six CDRs in the variable regions ofthe heavy chain and light chain interact and form the antigen-bindingdomain of the antibody. On the other hand, where there is only onesingle CDR, while the antigen-binding affinity is known to be lower ascompared to where six CDRs are present, it has still the ability torecognize and bind to the antigen.

Ig antibodies are classified into several classes (isotypes) based onstructural differences in their constant regions. In many mammals, theyare categorized into five immunoglobulin classes based on structuraldifferences in the constant region: IgG, IgA, IgM, IgD, and IgE.Furthermore, in the case of human, IgG has four subclasses: IgG1, IgG2,IgG3, and IgG4; and IgA has two subclasses: IgA1 and IgA2. The heavychain is classified into γ chain, μ chain, α chain, δ chain, and ε chainaccording to differences in the constant region, and based on thesedifferences, there are five immunoglobulin classes (isotypes): IgG, IgM,IgA, IgD, and IgE. On the other hand, there are two types of lightchains: λ chain and κ chain, and all immunoglobulins have either ofthese two.

In one embodiment, where an antibody of Disclosure A or B has a heavychain, for example, the heavy chain may be any one of γ chain, μ chain,α chain, δ chain, and ε chain, or may be derived from any one of them,and where an antibody of Disclosure A or B has a light chain, forexample, the light chain may be either κ chain or λ chain, or may bederived from either. Furthermore, within the scope of Disclosures A andB described herein, the antibody may be of any isotype (for example,IgG, IgM, IgA, IgD, or IgE) and of any subclass (for example, humanIgG1, IgG2, IgG3, IgG4, IgA1, and IgA2; mouse IgG1, IgG2a, IgG2b, andIgG3), or may be derived from any one of them, but is not limitedthereto.

Within the scope of Disclosures A and B described herein, an“antigen-binding domain” may have any structure as long as it binds toan antigen of interest. Such domains may include, for example, thevariable regions of antibody heavy chains and light chains (for example,1 to 6 CDRs); a module of about 35 amino acids referred to as A domain,which is contained in Avimer, a cell membrane protein present in thebody (WO2004/044011 and WO2005/040229); Adnectin containing the 10Fn3domain which binds to the protein in the glycoprotein fibronectinexpressed on cell membrane (WO2002/032925); Affibody, having as scaffoldthe IgG-binding domain constituting a three-helix bundle of 58 aminoacids of Protein A (WO1995/001937); Designed Ankyrin Repeat Proteins(DARPins) which are a region exposed on the molecular surface of anAnkyrin repeat (AR) having a structure in which a subunit with a turncontaining 33 amino acid residues, two antiparallel helices, and a loopis repeatedly stacked (WO2002/020565); Anticalins and others, which area four loop region supporting one side of a centrally-twisted barrelstructure of eight antiparallel strands that are highly conserved amonglipocalin molecules such as neutrophil gelatinase-associated lipocalin(NGAL) (WO2003/029462); and the concave region formed by theparallel-sheet structure inside the horseshoe-shaped structure formed bystacked repeats of the leucine-rich-repeat (LRR) module of the variablelymphocyte receptor (VLR) which does not have a immunoglobulin structureand is used in the system of acquired immunity in jawless vertebratessuch as lamprey and hagfish (WO2008/016854). Preferred antigen-bindingdomains of Disclosure A or B may include those having IgG antibodyheavy-chain and light-chain variable regions, and more specifically,ScFv, single chain antibodies, Fv, scFv₂ (single chain Fv₂), Fab, andF(ab′)₂.

In one embodiment of Disclosure A, “ion concentration” is notparticularly limited and refers to hydrogen ion concentration (pH) ormetal ion concentration. Herein, “metal ions” can be any one of ions ofgroup I elements except hydrogen, such as alkaline metals and coppergroup elements, group II elements such as alkaline earth metals and zincgroup elements, group III elements except boron, group IV elementsexcept carbon and silicon, group VIII elements such as iron group andplatinum group elements, elements belonging to subgroup A of groups V,VI, and VII, and metal elements such as antimony, bismuth, and polonium.Metal atoms have the property of releasing valence electrons to becomecations. This is referred to as ionization tendency. Metals with strongionization tendency are assumed to be chemically active.

In one embodiment of Disclosure A, preferred metal ions may be calciumion, as described in detail in WO2012/073992 and WO2013/125667.

In one embodiment of Disclosure A, “ion concentration condition(s)” maybe a condition that focuses on differences in the biological behavior ofan ion concentration-dependent antibody between a low ion concentrationand a high ion concentration. Furthermore, “the antigen-binding activitychanges according to the ion concentration condition” can mean that theantigen-binding activity of an ion concentration-dependentantigen-binding domain or an ion concentration-dependent antibody ofDisclosure A or B changes between a low ion concentration and a high ionconcentration. Such cases include, for example, those with higher(stronger) or lower (weaker) antigen-binding activity at a high ionconcentration than at a low ion concentration, without being limitedthereto.

In one embodiment of Disclosure A, the ion concentration can be hydrogenion concentration (pH) or calcium ion concentration. Where the ionconcentration is hydrogen ion concentration (pH), the ionconcentration-dependent antigen-binding domain may also be referred toas a “pH-dependent antigen-binding domain”; and where the ionconcentration is calcium ion concentration, it may also be referred toas a “calcium ion concentration-dependent antigen-binding domain”.

In one embodiment in the context of Disclosure A, the ionconcentration-dependent antigen-binding domains, ionconcentration-dependent antibodies, ion concentration-dependentantigen-binding domains with increased pI, and ionconcentration-dependent antibodies with increased pI can be obtainedfrom libraries primarily consisting of antibodies that differ insequence (have variability) and whose antigen-binding domains contain atleast one amino acid residue that causes a change in the antigen-bindingactivity of the antigen-binding domain or antibody according to the ionconcentration condition. The antigen-binding domains may be preferablylocated within the light chain variable region (which may be modified)and/or the heavy chain variable region (which may be modified).Furthermore, to construct a library, such light-chain or heavy-chainvariable regions may be combined with heavy-chain or light-chainvariable regions constructed as a randomized variable region sequencelibrary. Where the ion concentration is hydrogen or calcium ionconcentration, non-limiting examples of the library include, forexample, libraries in which heavy chain variable regions constructed asa randomized variable region sequence library are combined with lightchain variable region sequences in which amino acid residue(s) in a germline sequence such as SEQ ID NO:1 (Vk1), SEQ ID NO:2 (Vk2), SEQ ID NO:3(Vk3), or SEQ ID NO:4 (Vk4) has been substituted with at least one aminoacid residue that can alter the antigen-binding activity depending onion concentrations. Furthermore, where the ion concentration is calciumion concentration, the library includes, for example, those in which theheavy chain variable region sequence of SEQ ID NO:5 (6RL#9-IgG1) or SEQID NO:6 (6KC4-1#85-IgG1) is combined with light chain variable regionsconstructed as a randomized variable region sequence library or lightchain variable regions having a germ line sequence.

In one embodiment, where the ion concentration is calcium ionconcentration, the high calcium ion concentration is not particularlylimited to a specific value; however, the concentration may be selectedbetween 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3mM, between 200 μM and 2 mM, or between 400 μM and 1 mM. A concentrationselected between 500 μM and 2.5 mM, which is close to the plasma (blood)concentration of calcium ion in vivo, may be also preferred. The lowcalcium ion concentration is not particularly limited to a specificvalue; however, the concentration may be selected between 0.1 μM and 30between 0.2 μM and 20 between 0.5 and 10 or between 1 μM and 5 orbetween 2 μM and 4 μM. A concentration selected between 1 μM and 5 whichis close to the concentration of calcium ion in early endosomes in vivo,may be also preferred.

Whether the antigen-binding activity of an antigen-binding domain orantibody containing the domain changes according to the metal ionconcentration (for example, calcium ion concentration) condition can bereadily determined by known methods, for example, by the methodsdescribed herein in the context of Disclosure A, or described inWO2012/073992. For example, the antigen-binding activity of anantigen-binding domain or antibody containing the domain can be measuredat low and high calcium ion concentrations and compared. In this case,conditions other than the calcium ion concentration may be preferablythe same. Furthermore, conditions other than the calcium ionconcentration in determining the antigen-binding activity can beappropriately selected by those of ordinary skill in the art. Theantigen-binding activity can be determined, for example, under theconditions of HEPES buffer at 37° C., or using the BIACORE (GEHealthcare) or others.

In one embodiment in the context of Disclosure A, it is preferable thatthe antigen-binding activity of the ion concentration-dependentantigen-binding domain, ion concentration-dependent antibody, ionconcentration-dependent antigen-binding domain with increased pI, or ionconcentration-dependent antibody with increased pI is higher under ahigh calcium ion concentration condition than under a low calcium ionconcentration condition. In this case, the ratio between theantigen-binding activity under a low calcium ion concentration conditionand the antigen-binding activity under a high calcium ion concentrationcondition is not limited; however, the value of the ratio of the KD(dissociation constant) for an antigen under a low calcium ionconcentration condition to the KD under a high calcium ion concentrationcondition, i.e., KD (3 Ca)/KD (2 mM Ca), may be preferably 2 or more,more preferably 10 or more, and still more preferably 40 or more. Theupper limit of the KD (3 μM Ca)/KD (2 mM Ca) value is not limited, andmay be any value such as 400, 1000, or 10000.

Where the antigen is a soluble antigen, the dissociation constant (KD)can be used as the value for antigen-binding activity. Meanwhile, wherethe antigen is a membrane antigen, the apparent dissociation constant(KD) can be used. The dissociation constant (KD) and apparentdissociation constant (KD) can be determined by known methods, forexample, by BIACORE (GE healthcare), Scatchard plot, or flow cytometer.

Alternatively, for example, the dissociation rate constant (kd) can alsobe used as another indicator to represent the binding activity ratio.Where the dissociation rate constant (kd) is used instead of thedissociation constant (KD) as an indicator to represent the antigenbinding activity ratio, the value of the ratio of thelow-calcium-ion-concentration-condition dissociation rate constant (kd)to the high-calcium-ion-concentration-condition dissociation rateconstant (kd), i.e., kd (low calcium ion concentration condition)/kd(high calcium ion concentration condition), may be preferably 2 or more,more preferably 5 or more, still more preferably 10 or more, and yetmore preferably 30 or more. The upper limit of the kd (low calcium ionconcentration condition)/kd (high calcium ion concentration condition)value is not limited, and may be any value such as 50, 100, or 200.

Where the antigen is a soluble antigen, the dissociation rate constant(kd) can be used as the value for antigen-binding activity. Meanwhile,where the antigen is a membrane antigen, the apparent dissociation rateconstant (kd) can be used. The dissociation rate constant (kd) and theapparent dissociation rate constant (kd) can be determined by knownmethods, for example, by BIACORE (GE healthcare) or flow cytometer.

In one embodiment, methods for producing or screening for calcium ionconcentration-dependent antigen-binding domains or calcium ionconcentration-dependent antibodies whose antigen-binding activity ishigher at a high calcium ion concentration condition than at a lowcalcium ion concentration condition, or libraries thereof, are notlimited. The methods include, for example, those described inWO2012/073992 (for example, paragraphs 0200-0213).

Such a method may comprise, for example:

-   (a) determining the antigen-binding activity of an antigen-binding    domain or antibody at a low calcium ion concentration condition;-   (b) determining the antigen-binding activity of an antigen-binding    domain or antibody at a high calcium ion concentration condition;    and-   (c) selecting an antigen-binding domain or antibody whose    antigen-binding activity at a low calcium ion concentration    condition is lower than the antigen-binding activity at a high    calcium ion concentration condition.

Alternatively, the method may comprise, for example:

-   (a) contacting an antigen with an antigen-binding domain or    antibody, or a library thereof, at a high calcium ion concentration    condition;-   (b) incubating an antigen-binding domain or antibody that bound to    the antigen in step (a) at a low calcium ion concentration    condition; and-   (c) isolating an antigen-binding domain or antibody that dissociated    in step (b).

Alternatively, the method may comprise, for example:

-   (a) contacting an antigen with an antigen-binding domain or    antibody, or a library thereof at a low calcium ion concentration    condition;-   (b) selecting an antigen-binding domain or antibody that does not    bind to the antigen or has a low antigen-binding ability in step    (a);-   (c) allowing the antigen-binding domain or antibody selected in    step (b) to bind to the antigen at a high calcium ion concentration    condition; and-   (d) isolating the antigen-binding domain or antibody that bound to    the antigen in step (c).

Alternatively, the method may comprise, for example:

-   (a) contacting an antigen-binding domain or antibody, or a library    thereof with an antigen-immobilized column at a high calcium ion    concentration condition;-   (b) eluting an antigen-binding domain or antibody bound to the    column in step (a) from the column at a low calcium ion    concentration condition; and-   (c) isolating an antigen-binding domain or antibody eluted in step    (b).

Alternatively, the method may comprise, for example:

-   (a) allowing an antigen-binding domain or antibody, or a library    thereof to pass through an antigen-immobilized column at a low    calcium ion concentration condition to collect an antigen-binding    domain or antibody eluted without binding to the column;-   (b) allowing an antigen-binding domain or antibody collected in    step (a) to bind to the antigen at a high calcium ion concentration    condition; and-   (c) isolating an antigen-binding domain or antibody bound to the    antigen in step (b).

Alternatively, the method may comprise, for example:

-   (a) contacting an antigen with an antigen-binding domain or    antibody, or a library thereof at a high calcium ion concentration    condition;-   (b) obtaining an antigen-binding domain or antibody bound to the    antigen in step (a);-   (c) incubating an antigen-binding domain or antibody obtained in    step (b) at a low calcium ion concentration; and-   (d) isolating an antigen-binding domain or antibody whose    antigen-binding activity in step (c) is weaker than the criterion    selected in step (b).

Each step of these various screening methods may be repeated severaltimes, or the steps may be combined appropriately to obtain the mostsuitable molecules. The aforementioned conditions may be suitablyselected for the low and high calcium ion concentration conditions.Desired calcium ion concentration-dependent antigen-binding domains orcalcium ion concentration-dependent antibodies can be obtained thereby.

In the context of Disclosure A, in one embodiment, the antigen-bindingdomains or antibodies as a starting material may be, for example,modified antigen-binding domains or antibodies that have an increased pIas a result of modifying the charge of at least one amino acid residuethat can be exposed on their surface. In an alternative embodiment,where amino acids that change the binding activity of an ionconcentration-dependent antigen-binding domain are introduced into thesequence, they may be introduced in conjunction with a chargemodification of at least one amino acid residue that can be exposed onthe surface of the antigen-binding domain or antibody so as to increasethe pI.

Alternatively, in the context of present invention A, for example, it ispossible to use pre-existing antigen-binding domains or antibodies,preexisting libraries (phage library, etc.); antibodies prepared fromhybridomas obtained by immunizing animals or from B cells of immunizedanimals, or libraries thereof; or antigen-binding domains, antibodies,or libraries obtained by introducing natural or unnatural amino acidmutations capable of chelating calcium (described below) thereinto (forexample, libraries with an increased content of calcium-chelatable aminoacids, or libraries introduced with calcium-chelatable amino acids atspecific sites).

In one embodiment in the context of Disclosure A, where the ionconcentration is calcium ion concentration, there is no limitation as tothe type of amino acids that change the binding activity of ionconcentration-dependent antigen-binding domains or ionconcentration-dependent antigen-binding domains with increased pI, aslong as they can form a calcium-binding motif. For example,calcium-binding motifs are known to those of ordinary skill in the art(for example, Springer et al. (Cell 102:275-277 (2000)); Kawasaki et al.(Protein Prof 2:305-490 (1995)); Moncrief et al. (J. Mol. Evol.30:522-562 (1990)); Chauvaux et al. (Biochem. J. 265:261-265 (1990));Bairoch et al. (FEBS Lett. 269:454-456 (1990)); Davis (New Biol.2:410-419 (1990)); Schaefer et al. (Genomics 25:638-643 (1995));Economou et al. (EMBO J. 9:349-354 (1990)); Wurzburg et al. (Structure.14(6):1049-1058 (2006)). Thus, where an antigen-binding domain has anarbitrary calcium-binding motif such as of a C-type lectin, for example,ASGPR, CD23, MBR, or DC-SIGN, the antigen-binding activity of the domaincan be changed according to the calcium ion concentration condition.Such calcium-binding motifs may include, for example, in addition tothose described above, the calcium-binding motif included in theantigen-binding domain shown in SEQ ID NO:7 (which corresponds to“Vk5-2”).

In one embodiment in the context of Disclosure A, where the ionconcentration is calcium ion concentration, amino acids having ametal-chelating activity may be used as amino acids that change thebinding activity of ion concentration-dependent antigen-binding domainsor ion concentration-dependent antigen-binding domains of with increasedpI. For example, any amino acids can be appropriately used as aminoacids having a metal-chelating activity, as long as they can form acalcium-binding motif. Specifically, such amino acids include thosehaving an electron-donating property. The amino acids preferablyinclude, but are not limited to, Ser (S), Thr (T), Asn (N), Gln (Q), Asp(D), and Glu (E).

The location of such amino acids having a metal-chelating activity in anantigen-binding domain is not limited to specific positions. In oneembodiment, the amino acids may be located at any positions in the heavychain variable region and/or light chain variable region that may forman antigen-binding domain. At least one amino acid residue that causescalcium ion concentration-dependent changes in the antigen-bindingactivity of an antibody may be contained, for example, in CDR (one ormore of CDR1, CDR2, and CDR3) and/or FR (one or more of FR1, FR2, FR3,and FR4) of the heavy chain and/or light chain. The amino acidresidue(s) may be placed, for example, at one or more of positions 95,96, 100a, and 101 according to Kabat numbering in heavy-chain CDR3; atone or more of positions 30, 31, and 32 according to Kabat numbering inlight-chain CDR1; at position 50 according to Kabat numbering inlight-chain CDR2; and/or at position 92 according to Kabat numbering inlight-chain CDR3. Those amino acid residues may be placed alone or incombination.

Troponin C, calmodulin, parvalbumin, myosin light chain, and others areknown to have multiple calcium-binding sites and assumed to be derivedfrom a common origin in molecular evolution, and in one embodiment, oneor more of light chain CDR1, CDR2, and CDR3 can be designed to containbinding motifs thereof. For the purpose described above, for example,the cadherin domain; the EF hand contained in calmodulin; the C2 domaincontained in Protein kinase C; the Gla domain contained inblood-clotting protein Factor IX; C-type lectin of theasialoglycoprotein receptor or mannose-binding receptor; the A domaincontained in the LDL receptor; Annexin; thrombospondin type-3 domain;and EGF-like domain may be suitably used.

In one embodiment, where the ion concentration is hydrogen ionconcentration (pH), the concentration condition of proton, i.e., nucleusof a hydrogen atom, is used synonymously with the condition of hydrogenindex (pH). Where the amount of activity of hydrogen ion in an aqueoussolution is represented by aH⁺, pH is defined as −log 10aH⁺. Where theionic strength of the aqueous solution is low (for example, less than10⁻³), aH⁺ is nearly equal to the hydrogen ion strength. For example,the ionic product for water at 25° C. and 1 atmosphere isKw=aH⁺*aOH=10⁻¹⁴; thus, for pure water, aH⁺=aOH=10⁻⁷. In this case, pH=7is neutral, and an aqueous solution with a pH of less than 7 is acidic,and an aqueous solution with a pH of greater than 7 is alkaline. Thus,the hydrogen ion concentration condition may be conditions that focus ondifferences in the biological behavior of a pH-dependent antibody at ahigh hydrogen ion concentration (acidic pH range) and at a low hydrogenion concentration (neutral pH range) for the hydrogen ion concentrationcondition or pH condition. For example, in the context of Disclosure A,“the antigen-binding activity at a high hydrogen ion concentration(acidic pH range) condition is lower than the antigen-binding activityat a low hydrogen ion concentration (neutral pH range) condition” canmean that the antigen-binding activity of an ion concentration-dependentantigen-binding domain, an ion concentration-dependent antibody, an ionconcentration-dependent antigen-binding domain with increased pI, or anion concentration-dependent antibody with increased pI is weaker at a pHselected from pH 4.0 to pH 6.5, preferably from pH 4.5 to pH 6.5, morepreferably from pH 5.0 to pH 6.5, and still more preferably from pH 5.5to pH 6.5, than at a pH selected from pH 6.7 to pH 10.0, preferably frompH 6.7 to pH 9.5, more preferably from pH 7.0 to pH 9.0, and still morepreferably from pH 7.0 to pH 8.0. Preferably, the above expression canmean that the antigen-binding activity at the pH within early endosomesin vivo is weaker than that at the plasma pH in vivo; and specificallycan mean that the antigen-binding activity of an antibody, for example,at pH 5.8 is weaker than that, for example, at pH 7.4.

Whether the antigen-binding activity of an antigen-binding domain or anantibody containing the domain changes according to the hydrogen ionconcentration condition can be readily assessed by known methods, forexample, by the assay methods described herein in the context ofDisclosure A, or described in WO2009/125825. For example, theantigen-binding activity of an antigen-binding domain or an antibodycontaining the domain toward an antigen of interest may be measured atlow and high hydrogen ion concentrations and compared. In this case, itis preferable that conditions other than the hydrogen ion concentrationare the same. Where determining the antigen-binding activity, those ofordinary skill in the art can suitably select conditions other than thehydrogen ion concentration, and for example, measurements can be carriedout under the condition of HEPES buffer at 37° C., or using the BIACORE(GE Healthcare), or the like.

Within the scope of Disclosure A described herein, unless particularlyspecified otherwise in the context, “neutral pH range” (also referred toas “low hydrogen ion concentration”, “high pH”, “neutral pH condition”,or “neutral pH”) is not particularly limited to a specific value;however, it may be preferably selected from pH 6.7 to pH 10.0, from pH6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0. Theneutral pH range may be preferably pH 7.4 which is close to the in vivopH in plasma (blood), but for the convenience of measurement, forexample, pH 7.0 may be used.

Within the scope of Disclosure A described herein, unless particularlyspecified otherwise in the context, “acidic pH range” (also referred toas “high hydrogen ion concentration”, “low pH”, “acidic pH condition”,or “acidic pH”) is not particularly limited to a specific value;however, it may be preferably selected from pH 4.0 to pH 6.5, from pH4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. The acidic pHrange may be preferably pH 5.8 which is close to the in vivo hydrogenion concentration in the early endosome, but for the convenience ofmeasurement, for example, pH 6.0 may be used.

In one embodiment in the context of Disclosure A, where the ionconcentration is hydrogen ion concentration, it is preferable that theantigen-binding activity of the ion concentration-dependentantigen-binding domain, ion concentration-dependent antibody, ionconcentration-dependent antigen-binding domain with increased pI, or ionconcentration-dependent antibody with increased pI is higher under aneutral pH condition than under an acidic pH condition. In this case,the ratio of the antigen-binding activity under a neutral pH conditionto the antigen-binding activity under an acidic pH condition is notlimited; however, the value of the ratio of the dissociation constant(KD) for an antigen at an acidic pH condition to the KD at a neutral pHcondition, i.e., KD (acidic pH range)/KD (neutral pH range), (forexample, KD (pH 5.8)/KD (pH 7.4)) may be 2 or more; 10 or more; or 40 ormore. The upper limit of KD (acidic pH range)/KD (neutral pH range)value is not limited, and may be any value such as 400, 1000, or 10000.

In an alternative embodiment, it is also possible to use, for example,the dissociation rate constant (kd) as an indicator to represent theabove binding activity ratio. Where the dissociation rate constant (kd)is used instead of the dissociation constant (KD) as an indicator torepresent the binding activity ratio, the value of the ratio of thedissociation rate constant (kd) for an antigen at a high hydrogen ionconcentration condition to that at a low hydrogen ion concentrationcondition, i.e., kd (acidic pH range)/kd (neutral pH range) may be 2 ormore, 5 or more, 10 or more, or 30 or more. The upper limit of the kd(acidic pH range)/kd (neutral pH range) value is not limited, and may beany value such as 50, 100, or 200.

Where the antigen is a soluble antigen, the value of the antigen-bindingactivity can be represented by the dissociation rate constant (kd),whereas where the antigen is a membrane antigen, such value can berepresented by the apparent dissociation rate constant (apparent kd).The dissociation rate constant (kd) and apparent dissociation rateconstant (apparent kd) can be determined by known methods, for example,by using the BIACORE (GE healthcare) or a flow cytometer.

In one embodiment, methods for producing or screening for pH-dependentantigen-binding domains or pH-dependent antibodies whose antigen-bindingactivity is higher under a neutral pH condition than under an acidic pHcondition, or libraries thereof, are not limited. Such methods include,for example, those described in WO2009/125825 (for example, paragraphs0158-0190).

Such a method may comprise, for example:

-   (a) determining the antigen-binding activity of an antigen-binding    domain or antibody in an acidic pH condition;-   (b) determining the antigen-binding activity of an antigen-binding    domain or antibody in a neutral pH condition; and-   (c) selecting an antigen-binding domain or antibody whose    antigen-binding activity is lower in the acidic pH condition than in    the neutral pH condition.

Alternatively, the method may comprise, for example:

-   (a) contacting an antigen with an antigen-binding domain or    antibody, or a library thereof, in a neutral pH condition;-   (b) incubating an antigen-binding domain or antibody bound to the    antigen in step (a) in an acidic pH condition; and-   (c) isolating an antigen-binding domain or antibody that dissociated    in step (b).

Alternatively, the method may comprise, for example:

-   (a) contacting an antigen with an antigen-binding domain or    antibody, or a library thereof in an acidic pH condition;-   (b) selecting an antigen-binding domain or antibody that does not    bind to the antigen or has a low antigen-binding ability in step    (a);-   (c) allowing the antigen to bind to the antigen-binding domain or    antibody selected in step (b) in a neutral pH condition; and-   (d) isolating an antigen-binding domain or antibody that bound to    the antigen in step (c).

Alternatively, the method may comprise, for example:

-   (a) contacting an antigen-binding domain or antibody, or a library    thereof with an antigen-immobilized column in a neutral pH    condition;-   (b) eluting an antigen-binding domain or antibody bound to the    column in step (a) from the column in an acidic pH condition; and-   (c) isolating an antigen-binding domain or antibody eluted in step    (b).

Alternatively, the method may comprise, for example:

-   (a) allowing an antigen-binding domain or antibody, or a library    thereof to pass through an antigen-immobilized column in an acidic    pH condition to collect an antigen-binding domain or antibody eluted    without binding to the column;-   (b) allowing an antigen-binding domain or antibody collected in    step (a) to bind to the antigen in a neutral pH condition; and-   (c) isolating an antigen-binding domain or antibody bound to the    antigen in step (b).

Alternatively, the method may comprise, for example:

-   (a) contacting an antigen with an antigen-binding domain or    antibody, or a library thereof in a neutral pH condition;-   (b) obtaining an antigen-binding domain or antibody bound to the    antigen in step (a);-   (c) incubating an antigen-binding domain or antibody obtained in    step (b) in an acidic pH condition; and-   (d) isolating an antigen-binding domain or antibody whose    antigen-binding activity in step (c) is weaker than the criterion    selected in step (b).

Each step in these various screening methods may be repeated severaltimes, or the steps may be combined. The aforementioned conditions maybe suitably selected for the acidic and neutral pH conditions. DesiredpH-dependent antigen-binding domains or pH-dependent antibodies can beobtained thereby.

In the context of Disclosure A, in one embodiment, the antigen-bindingdomains or antibodies as a starting material may be, for example,modified antigen-binding domains or antibodies that have an increased pIas a result of modifying the charge of at least one amino acid residuethat can be exposed on their surface. In an alternative embodiment,where amino acids that change the binding activity of an ionconcentration-dependent antigen-binding domain are introduced into thesequence, they may be introduced in conjunction with a chargemodification of at least one amino acid residue that can be exposed onthe surface of the antigen-binding domain or antibody so as to increasethe pI.

Alternatively, in the context of present invention A, for example, it ispossible to use pre-existing antigen-binding domains or antibodies,pre-existing libraries (phage library, etc.); antibodies prepared fromhybridomas obtained by immunizing animals or from B cells of immunizedanimals, or libraries thereof; or antigen-binding domains, antibodies,or libraries obtained by introducing natural or unnatural amino acidmutations having a side-chain with a pKa of 4.0-8.0 (described below)thereinto (for example, libraries with an increased content of naturalor unnatural amino acid mutations with a side-chain pKa of 4.0-8.0, orlibraries introduced at specific sites with natural or unnatural aminoacid mutations with a side-chain pKa of 4.0-8.0). Such a preferredantigen-binding domain can have, for example, an amino acid sequence inwhich at least one amino acid residue has been substituted with an aminoacid(s) with a side-chain pKa of 4.0-8.0 and/or which has been insertedwith amino acid(s) with a side-chain pKa of 4.0-8.0, as described inWO2009/125825.

In one embodiment in the context of Disclosure A, the site at which themutation of amino acids with a side-chain pKa of 4.0-8.0 is introducedis not limited, and the mutation may be introduced at any site as longas the antigen-binding activity becomes weaker in an acidic pH rangethan in a neutral pH range (the KD (acidic pH range)/KD (neutral pHrange) value is increased or the kd (acidic pH range)/kd (neutral pHrange) value is increased) as compared to before substitution orinsertion. Where the antibody has a variable region or CDR(s), the sitemay be within the variable region or CDR(s). The number of amino acidsthat are substituted or inserted can be appropriately determined bythose of ordinary skill in the art; and the number may be one or more.Furthermore, it is possible to delete, add, insert, and/or substitute,or modify other amino acids in addition to the substitution or insertiondescribed above. Substitution with or insertion of amino acids with aside-chain pKa of 4.0-8.0 may be carried out in a random fashion byscanning methods such as histidine scanning, in which histidine is usedinstead of alanine in alanine scanning known to those of ordinary skillin the art, and/or antibodies whose KD (acidic pH range)/KD (neutral pHrange) value or kd (acidic pH range)/kd (neutral pH range) value hasincreased as compared to before mutation may be selected from among theantigen-binding domains or antibodies that result from randomsubstitution with or insertion mutations of these amino acids, orlibraries thereof.

Furthermore, the antigen-binding domains or antibodies may be preferablythose whose antigen-binding activity in a neutral pH range before andafter these mutations is not significantly reduced, is not substantiallyreduced, is substantial identical, or is increased; and in other words,those whose activity may be maintained at at least 10% or higher,preferably 50% or higher, still more preferably 80% or higher, and yetmore preferably 90% or higher, or even higher. Where the bindingactivity of an antigen-binding domain or antibody is decreased due tosubstitution with or insertion of amino acids with a pKa of 4.0-8.0, thebinding activity may be recovered or increased by e.g., substituting,deleting, adding, or inserting one or more amino acids at sites otherthan the substitution or insertion sites described above.

In an alternative embodiment, amino acids with a side chain pKa of4.0-8.0 may be placed at any location within the heavy-chain and/orlight-chain variable regions that may form an antigen-binding domain. Atleast one amino acid residue with a side-chain pKa of 4.0-8.0 may belocated, for example, in the CDR (one or more of CDR1, CDR2, and CDR3)and/or FR (one or more of FR1, FR2, FR3, and FR4) of the heavy chainand/or light chain. Such amino acid residues include, but are notlimited to, amino acid residues at one or more of positions 24, 27, 28,31, 32, and 34 according to Kabat numbering in the light-chain variableregion CDR1; amino acid residues at one or more of positions 50, 51, 52,53, 54, 55, and 56 according to Kabat numbering in the light-chainvariable region CDR2; and/or amino acid residues at one or more ofpositions 89, 90, 91, 92, 93, 94, and 95A according to Kabat numberingin the light-chain variable region CDR3. Those amino acid residues maybe included alone or in combination, as long as the antigen-bindingactivity of the antibody changes according to the hydrogen ionconcentration condition.

In one embodiment within the scope of Disclosure A, an arbitrary aminoacid residue can be suitably used as the amino acid residue that changesthe antigen-binding activity of the antigen-binding domain or antibodyaccording to the hydrogen ion concentration condition. Specifically,such amino acid residues can include those with a side-chain pKa of4.0-8.0. Such amino acids having an electron-donating property mayinclude, for example, natural amino acids such as His (H) and Glu (E),and unnatural amino acids such as histidine analogs (US2009/0035836),m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38)(Heyl et al., Bioorg. Med. Chem. 11(17):3761-3768 (2003)). The aminoacid residues may preferably include, for example, amino acids with aside-chain pKa of 6.0-7.0, and in particular His (H).

Within the scope of Disclosure A described herein, unless otherwisespecified and unless there are inconsistencies in the context, it isunderstood that the isoelectric point (pI) may be either a theoreticalor an experimentally determined isoelectric point, and it is alsoreferred to as “pI”.

The pI value can be determined experimentally, for example, byisoelectric focusing electrophoresis. Meanwhile, the theoretical pIvalue can be calculated using gene and amino acid sequence analysissoftware (Genetyx, etc.).

In one embodiment, whether the pI of an antibody with increased pI or anantibody of Disclosure A has been increased as compared to the antibodybefore modification (a native antibody (for example, a native Igantibody, preferably a native IgG antibody) or reference antibody (e.g.,an antibody before antibody modification, or prior to or during libraryconstruction)) can be determined by carrying out, in addition to orinstead of the above-described methods, antibody pharmacokinetics testusing plasma, for example, from mice, rats, rabbits, dogs, monkeys, orhumans, in combination with methods such as BIACORE, cell proliferationassay, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA), orfluorescent immunoassay.

Within the scope of Disclosure A described herein, an “amino acidresidue that can be exposed on the surface” generally can refer to anamino acid residue located on the surface of a polypeptide constitutingan antibody. An “amino acid residue located on the surface of apolypeptide” can refer to an amino acid residue whose side chain can bein contact with solvent molecules (which in general may be mostly watermolecules). However, the side chain does not necessarily have to bewholly in contact with solvent molecules, and when even a portion of theside chain is in contact with the solvent molecules, the amino acidresidue is defined as an “amino acid located on the surface”. The aminoacid residues located on the surface of a polypeptide can also includeamino acid residues located close to the antibody surface and therebycan have a mutual electric charge influence from other amino acidresidue(s) whose side chain, even partly, is in contact with the solventmolecules. Those of ordinary skill in the art can prepare a homologymodel of a polypeptide or antibody by for example homology modelingusing commercially available softwares. Alternatively, it is possible touse methods such as X-ray crystallography. The amino acid residues thatmay be exposed on the surface can be determined, for example, usingcoordinates from a three-dimensional model of an antibody using acomputer program such as InsightII program (Accelrys). Surface-exposedsites may be determined using algorithms known in the technical field(for example, Lee and Richards (J. Mol. Biol. 55:379-400 (1971));Connolly (J. Appl. Cryst. 16:548-558 (1983)). Surface-exposable sitescan be determined using software suitable for protein modeling andthree-dimensional structure information obtained from the antibody.Software available for such purposes includes, for example, the SYBYLBiopolymer Module software (Tripos Associates). When an algorithmrequires a user input size parameter, the “size” of a probe used in thecalculation may be set to about 1.4 Angstrom (Å) or less in radius.Furthermore, methods for determining surface-exposed regions and areasusing software for personal computers have been described by Pacios(Pacios, Comput. Chem 18(4):377-386 (1994); J. Mol. Model. 1:46-53(1995)). Based on such information as described above, appropriate aminoacid residues located on the surface of a polypeptide that constitutesan antibody can be selected.

A method for increasing the pI of a protein is, for example, to reducethe number of amino acids with a negatively charged side chain at aneutral pH condition (for example, aspartic acid and glutamic acid)and/or to increase the number of amino acids with a positively chargedside chain (for example, arginine, lysine and histidine). Amino acidresidues with a negatively charged side chain have a negative chargerepresented as −1 at a pH condition that is sufficiently higher thantheir side chain pKa, which is a theory well known to those of ordinaryskill in the art. For example, the theoretical pKa for the side chain ofaspartic acid is 3.9, and the side chain has a negative chargerepresented as −1 at a neutral pH condition (for example, in a solutionof pH 7.0). Conversely, amino acid residues with a positively chargedside chain have a positive charge represented as +1 at a pH conditionthat is sufficiently lower than their side chain pKa. For example, thetheoretical pKa for the side chain of arginine is 12.5, and the sidechain has a positive charge represented as +1 at a neutral pH condition(for example, in a solution of pH 7.0). Amino acid residues whose sidechain has no charge at a neutral pH condition (for example, in asolution of pH 7.0) are known to include 15 types of natural aminoacids, i.e., alanine, cysteine, phenylalanine, glycine, isoleucine,leucine, methionine, asparagine, proline, glutamine, serine, threonine,valine, tryptophan, and tyrosine. As a matter of course, it isunderstood that amino acids for changing the pI may be unnatural aminoacids.

From the above, as a method for increasing the pI of a protein at aneutral pH condition (for example, in a solution of pH 7.0), a chargealteration of +1 can be conferred to a protein of interest, for example,by substituting amino acids (residues) with non-charged side chains foraspartic acid (residue) or glutamic acid (residue) (whose side chain hasa negative charge of −1) in the amino acid sequence of the protein.Furthermore, a charge alteration of +1 can be conferred to the protein,for example, by substituting arginine or lysine (whose side chain has apositive charge of +1) for amino acid (residue) whose side chain has nocharge. Moreover, a charge alteration of +2 can be conferred at a timeto the protein by substituting arginine or lysine (whose side chain hasa positive charge of +1) for aspartic acid or glutamic acid (whose sidechain has a negative charge of −1). Alternatively, to increase the pI ofa protein, amino acids with a side chain having no charge and/or aminoacids having a positively charged side chain can be added or insertedinto the amino acid sequence of the protein, or amino acids with a sidechain having no charge and/or amino acids with a negatively charged sidechain present in the amino acid sequence of the protein can be deleted.It is understood that, for example, the N-terminal and C-terminal aminoacid residues of a protein have a main chain-derived charge (NH³⁺ of theamino group at the N-terminus and COO⁻ of the carbonyl group at theC-terminus) in addition to their side chain-derived charges. Thus, thepI of a protein can also be increased by performing to the mainchain-derived functional groups some addition, deletion, substitution,or insertion.

Those of ordinary skill in the art would appreciate that the effect ofchanging the net charge or pI of a protein, which is obtained bymodifying one or more amino acids (residues) in the amino acid sequencewith a focus on the presence or magnitude of electrical charges of theamino acids (residues), does not exclusively (or substantially) dependon the antibody-constituting amino acid sequences per se or the type oftarget antigen, but rather depends on the type and number of amino acidresidues that are added, deleted, substituted, or inserted.

Antibodies which have been modified to have an increased pI bymodification on at least one amino acid residue that can be exposed onthe antibody surface (“antibodies with increased pI” or “pI-increasedantibodies”) can be taken up more rapidly into cells or can promoteantigen elimination from the plasma, as described or suggested in, forexample, WO2007/114319, WO2009/041643, WO2014/145159, or WO2012/016227.

Of the several antibody isotypes, for example, the IgG antibody has asufficiently large molecular weight, and thus its major metabolicpathway is not through renal excretion. The IgG antibody, which has anFc region as a part of the molecule, is known to be recycled through asalvage pathway via FcRn, and thus has a long in vivo half-life. The IgGantibody is assumed to be mainly metabolized via a metabolic pathway inendothelial cells (He et al., J. Immunol. 160(2):1029-1035 (1998)).Specifically, it is believed that when taken up into endothelial cellsnonspecifically, IgG antibodies are recycled by binding to FcRn, whileIgG antibodies that could not bind are metabolized. The plasma half-lifeof an IgG antibody may be shortened when its Fc region is modified suchthat its FcRn-binding activity is reduced. On the other hand, the plasmahalf-life of an antibody with an increased pI has been demonstrated todepend on the pI in a highly correlated manner, as described in e.g.,WO2007/114319 and WO2009/041643. Specifically, the plasma half-life ofthe pI-increased antibodies described in the above documents was reducedwithout modifying the amino acid sequence constituting Fc which couldpotentially lead to acquisition of immunogenicity, and this resultsuggests that the pI-increasing technology is widely applicable even toany types of antibody molecules whose main metabolic pathway is renalexcretion, such as scFv, Fab, or Fc fusion proteins.

The pH concentration in biological fluids (for example, plasma) is in aneutral pH range. Without being bound by a particular theory, it isbelieved that in biological fluids, the net positive charge of apI-increased antibody is increased due to the increased pI, and as aresult the antibody is more strongly attracted by physicochemicalCoulomb interaction to the endothelial cell surface whose net charge isnegative, when compared to antibodies whose pI has not been increased;via non-specific binding, the antibody binds thereto and is taken upinto cells, which results in shortening of the antibody half-life inplasma or enhancement of antigen elimination from plasma. Furthermore,increasing the pI of an antibody enhances uptake into cells of theantibody (or antigen/antibody complex) and/or intracellularpermeability, which is considered to result in reducing the antibodyconcentration in plasma, reducing the antibody bioavailability, and/orshortening the antibody half-life in plasma; and these phenomena areexpected to occur commonly in vivo, regardless of cell type, tissuetype, organ type, etc. Furthermore, where an antibody forms a complexwith an antigen and is taken up into cells, not only the antibody's pIbut also the antigen's pI can have an influence on the decrease orincrease of the uptake into cells.

In one embodiment, methods for producing or screening for antibodieswith an increased pI may include, for example, those described inWO2007/114319 (for example, paragraphs 0060-0087), WO2009/041643 (forexample, paragraph 0115), WO2014/145159, and WO2012/016227. Such amethod may comprise, for example:

-   (a) modifying a nucleic acid that encodes an antibody comprising at    least one amino acid residue that can be exposed on the antibody    surface such that the charge of the amino acid residue(s) is    modified so as to increase the pI of the antibody;-   (b) culturing a host cell such that the nucleic acid is expressed;    and-   (c) collecting an antibody from the host cell culture.

Alternatively, the method may comprise, for example:

-   (a′) modifying a nucleic acid that encodes an antibody comprising at    least one amino acid residue that can be exposed on the antibody    surface such that the charge of the amino acid residue(s) is    modified;-   (b′) culturing a host cell such that the nucleic acid is expressed;-   (c′) collecting an antibody from the host cell culture; and-   (d′) (optionally confirming or measuring and) selecting an antibody    with a pI increased as compared to an antibody before the    modification. Here, the antibody as a starting material or the    antibody before the modification or the reference antibody may be,    for example, an ion concentration-dependent antibody. Alternatively,    when modifying the amino acid residue(s), amino acid(s) that change    the binding activity of the ion concentration-dependent    antigen-binding domain may also be included in the sequence.

Alternatively, the method may simply be a method that comprisesculturing the host cells obtained in step (b) or (b′) and collecting anantibody from the cell culture.

In an alternative embodiment, the method may be, for example, a methodfor producing a multispecific antibody that comprises a firstpolypeptide and a second polypeptide, and optionally a third polypeptideand a fourth polypeptide, which comprises:

-   (A) modifying nucleic acid(s) that encodes the first polypeptide    and/or the second polypeptide, and optionally the third polypeptide    and/or the fourth polypeptide, any one or more of which comprises at    least one amino acid residue that can be exposed on the polypeptide    surface such that the charge of the amino acid residue(s) is    modified so as to increase the antibody's pI;-   (B) culturing a host cell such that the nucleic acid is expressed;    and-   (C) collecting a multispecific antibody from the host cell culture.

Alternatively, the method may comprise, for example:

-   (A′) modifying nucleic acid(s) that encodes the first polypeptide    and/or the second polypeptide, and optionally the third polypeptide    and/or the fourth polypeptide, any one or more of which comprises at    least one amino acid residue that can be exposed on the polypeptide    surface such that the charge of the amino acid residue(s) is    altered;-   (B′) culturing a host cell such that the nucleic acid is expressed;-   (C′) collecting a multispecific antibody from the host cell culture;    and-   (D′) (optionally confirming and) selecting an antibody whose pI is    increased as compared to an antibody before the modification.

Here, the antibody as a starting material or the antibody before themodification or the reference antibody may be, for example, an ionconcentration-dependent antibody. Alternatively, when modifying theamino acid residue(s), amino acid(s) that change the binding activity ofthe ion concentration-dependent antigen-binding domain may also beincluded in the sequence.

Alternatively, the method may simply be a method that comprisesculturing the host cells obtained in step (B) or (B′) and collecting anantibody from the cell culture. In this case, the polypeptides whosenucleic acid(s) is to be modified may be preferably a homomultimer ofthe first polypeptide, a homomultimer of the second polypeptide, or aheteromultimer of the first and second polypeptides (and optionally, ahomomultimer of the third polypeptide, a homomultimer of the fourthpolypeptide, or a heteromultimer of the third and fourth polypeptides).

In an alternative embodiment, the method may be, for example, a methodfor producing a humanized or human antibody with shortened half-life inplasma, which comprises: in an antibody which comprises CDR(s) selectedfrom the group consisting of human-derived CDR(s), CDR(s) derived froman animal other than human, and synthetic CDR(s); human-derived FR(s);and a human constant region, (I) modifying at least one amino acidresidue that can be exposed on the surface of at least one regionselected from the group consisting of the CDR(s), FR(s), and constantregion into amino acid residue(s) that has a different charge from theamino acid residue(s) present at the corresponding position(s) beforethe modification such that the pI of the antibody is increased.

Alternatively, the method may comprise, for example, in an antibodywhich comprises CDR(s) selected from the group consisting ofhuman-derived CDR(s), CDR(s) derived from an animal other than human,and synthetic CDR(s); human-derived FR(s); and a human constant region,

-   (I′) modifying at least one amino acid residue that can be exposed    on the surface of at least one region selected from the group    consisting of the CDR(s), FR(s), and constant region into amino acid    residue(s) that has a different charge from the amino acid    residue(s) present at the corresponding position(s) before the    modification; and-   (II′) (optionally confirming and) selecting an antibody whose pI is    increased as compared to an antibody before the modification.

Here, the antibody as a starting material or the antibody before themodification or the reference antibody may be, for example, an ionconcentration-dependent antibody. Alternatively, when modifying theamino acid residue(s), amino acid(s) that change the binding activity ofthe ion concentration-dependent antigen-binding domain may also beincluded in the sequence.

Alternatively, for example, it is possible to use pre-existingantigen-binding domains or antibodies, pre-existing libraries (phagelibrary, etc.); antibodies prepared from hybridomas obtained byimmunizing animals or from B cells of immunized animals, or librariesthereof; or antigen-binding domains or antibodies or libraries thereofwith increased pI, prepared by modifying, in the above-describedantigen-binding domains, antibodies, or libraries thereof, at least oneamino acid residue that can be exposed on the surface according to forexample any one of the above-described embodiments.

In one embodiment of the antibodies of Disclosure A, the pI value may bepreferably increased, for example, at least by 0.01, 0.03, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, or more, or at least by 0.6, 0.7, 0.8, 0.9, or more,and to significantly shorten the antibody half-life in plasma, the pIvalue may be increased, for example, by at least by 1.0, 1.1, 1.2, 1.3,1.4, 1.5, or more, or at least by 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, or more, or by 3.0 or more, as compared to the antibodiesbefore modification or alteration (native antibodies (for example,native Ig antibodies, preferably native IgG antibodies), or reference orparent antibodies (e.g., antibodies before antibody modification, orprior to or during library construction)). Those of ordinary skill inthe art can appropriately routinely determine the optimal pI value forthe antibodies of Disclosure A, in consideration of the balance betweentheir pharmacological effect and toxicity, and for example, the numberof antigen-binding domains of the antibodies or the pI of the antigenaccording to the purpose. Without being bound by a particular theory, itis believed that antibodies of Disclosure A, in one embodiment, arebeneficial because, in addition to the characteristic of being shuttledbetween plasma and cellular endosomes and repeated binding to multipleantigens with one single antibody molecule due to the presence of an ionconcentration-dependent antigen-binding domain, the antibody's netpositive charge is increased as a result of increase in pI and thisallows rapid cellular uptake of the antibody. These characteristicswould shorten the antibody half-life in plasma, increase theextracellular matrix-binding activity of the antibodies, or enhanceantigen elimination from plasma. One may decide on the optimal pI valueto take advantage of these characteristics.

In one embodiment in the context of Disclosure A, when compared to theantibodies before modification or alteration of at least one amino acidresidue to increase the pI (native antibodies (for example, native Igantibodies, preferably native IgG antibodies), or reference or parentantibodies (e.g., antibodies before antibody modification, or prior toor during library construction), which can be ionconcentration-dependent antibodies), the ion concentration-dependentantibodies of Disclosure A with increased pI may preferably enhanceantigen elimination from plasma, for example, by at least 1.1-fold,1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold,3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold,4.75-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold,8.5-fold, 9-fold, 9.5-fold, or 10-fold or more (when the antibodies areadministered in vivo), or their extracellular matrix-binding activitymay be preferably increased, for example, by at least 1.1-fold,1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold,3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold,4.75-fold, or 5-fold or more.

In one embodiment in the context of Disclosure A, when compared to theantibodies before introduction of an ion concentration-dependentantigen-binding domain (native antibodies (for example, native Igantibodies, preferably native IgG antibodies), or reference or parentantibodies (e.g., antibodies before antibody modification, or prior toor during library construction), which can be antibodies with anincreased pI), the ion concentration-dependent antibodies of DisclosureA with increased pI may preferably enhance antigen elimination fromplasma, for example, by at least 1.1-fold, 1.25-fold, 1.5-fold,1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold,3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold,5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold,9.5-fold, or 10-fold or more (when the antibodies are administered invivo), or their extracellular matrix-binding activity may be preferablyincreased, for example, by at least 1.1-fold, 1.25-fold, 1.5-fold,1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold,3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, or 5-foldor more.

In one embodiment, assay methods for assessing whether the extracellularmatrix-binding activity of antibodies of Disclosure A has been increasedas compared to the antibodies before modification or alteration (nativeantibodies (for example, native Ig antibodies, which can be native IgGantibodies), or reference or parent antibodies (e.g., antibodies beforeantibody modification, or prior to or during library construction),which can be ion concentration-dependent antibodies or antibodies withan increased pI) are not limited. For example, the assay can be carriedout using an ELISA system which detects the binding between an antibodyand an extracellular matrix, where an antibody is added to anextracellular matrix-immobilized plate, and a labeled antibody againstthe antibody is added thereto. Alternatively, as described in Examples 1to 4 herein and in WO2012/093704, it is also possible to useelectrochemiluminescence (ECL) which enables high sensitivity detectionof the extracellular matrix-binding ability. This method can beperformed, for example, using an ECL system in which a mixture of anantibody and a ruthenium antibody is added to an extracellularmatrix-immobilized plate and the binding between the antibodies and theextracellular matrix is measured based on the electrochemiluminescenceof ruthenium. The concentration of the antibody to be added can be setappropriately; the added concentration can be high in order to increasethe sensitivity for detecting extracellular matrix binding. Suchextracellular matrices may be derived from animals or plants, as long asthey contain glycoproteins such as collagen, proteoglycan, fibronectin,laminin, entactin, fibrin, and perlecan; and animal-derivedextracellular matrices may be preferred. For example, it is possible touse extracellular matrices derived from animals such as humans, mice,rats, monkeys, rabbits, or dogs. For example, a human-derived nativeextracellular matrix may be used as an indicator of antibodypharmacodynamics in human plasma. The condition for assessingextracellular matrix-binding of an antibody may be preferably a neutralpH range around pH 7.4, which is the physiological condition; however,the condition does not necessarily have to be a neutral range, and thebinding may also be assessed in an acidic pH range (for example, aroundpH 6.0). Alternatively, when assessing the extracellular matrix-bindingof an antibody, the assay can be performed in the co-presence of anantigen molecule to which the antibody binds and by assessing thebinding activity of the antigen-antibody complex toward theextracellular matrix.

In one embodiment, antibodies of Disclosure A (substantially) can retainthe antigen-binding activity when compared to the antibodies beforemodification or alteration of at least one amino acid residue toincrease pI (native antibodies (for example, native Ig antibodies,preferably native IgG antibodies) or reference antibodies (e.g.,antibodies before antibody modification, or prior to or during libraryconstruction)). In this case, “to (substantially) retain theantigen-binding activity” can mean to have an activity of at least 50%or more, preferably 60% or more, more preferably 70% or 75% or more, andstill more preferably 80%, 85%, 90%, or 95% or more as compared to thebinding activity of the antibodies before modification or alteration.Alternatively, antibodies of Disclosure A only need to retain bindingactivity to a degree that allows them to retain their functions whenthey bind to antigens; thus, the affinity determined at 37° C. under thephysiological conditions may be, for example, 100 nM or less, preferably50 nM or less, more preferably 10 nM or less, and still more preferably1 nM or less.

In one embodiment of Disclosure A, the expression of “modification of atleast one amino acid residue that can be exposed on the antibodysurface” or an equivalent expression can mean that one or more ofaddition, deletion, substitution and insertion are performed on at leastone amino acid residue that can be exposed on the surface of anantibody. Such modification may preferably include substitution of atleast one amino acid residue.

The substitution of amino acid residues can include, for example,substitution of amino acid residues whose side chain has no charge foramino acid residues having a negatively charged side chain, substitutionof amino acid residues having a positively charged side chain for aminoacid residues whose side chain has no charge, and substitution of aminoacid residues having a positively charged side chain for amino acidresidues having a negatively charged side chain in the amino acidsequence of an antibody of interest, which can be performed alone or inappropriate combinations. The insertion or addition of amino acidresidues can include, for example, insertion or addition of amino acidswhose side chain has no charge and/or insertion or addition of aminoacids having a positively charged side chain in the amino acid sequenceof an antibody of interest, which can be performed alone or inappropriate combinations. The deletion of amino acid residues caninclude, for example, deletion of amino acid residues whose side chainhas no charge and/or deletion of amino acid residues having a negativelycharged side chain in the amino acid sequence of an antibody ofinterest, which can be performed alone or in appropriate combinations.

Those of ordinary skill in the art can appropriately combine one of moreof these addition, deletion, substitution, and insertion in the aminoacid sequence of an antibody of interest. Modification that causes areduction in the local charge of amino acid residues is also acceptablesince the net pI of an antibody of Disclosure A only has to beincreased. For example, if desired, antibodies whose pI has beenincreased (too much) may be modified to decrease the pI (slightly). Itis also acceptable that the local charge of amino acid residues isdecreased as a result of modification of at least one amino acid residuecarried out simultaneously or at a different time for other purposes(for example, to increase antibody stability or to reduceimmunogenicity). Such antibodies include antibodies from librariesconstructed for specific purposes.

In one embodiment, among amino acids (residues) used for modifying atleast one amino acid residue that can be exposed on the antibodysurface, natural amino acids are as follows: an amino acid with anegatively charged side chain can be Glu (E) or Asp (D); an amino acidwhose side chain has no charge can be Ala (A), Asn (N), Cys (C), Gln(Q), Gly (G), His (H), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Ser(S), Thr (T), Trp (W), Tyr (Y), or Val (V); and an amino acid with apositively charged side chain can be His (H), Lys (K), or Arg (R).

As described in detail in Examples 1 to 4 herein, in a solution ofneutral pH (for example, pH 7.0), lysine and arginine have a positivecharge in almost 100% when present as a residue in an antibody, whilehistidine has a positive charge in only about 9% when present as aresidue in an antibody and the remaining major portion is assumed not tohave any charge. Thus, Lys (K) or Arg(R) is preferably selected as anamino acid with a positively charged side chain.

In one embodiment, antibodies of Disclosure A preferably has a variableregion and/or a constant region. Furthermore, the variable region maypreferably have a heavy chain variable region and/or a light chainvariable region, and/or may preferably have CDR(s) (for example, one ormore of CDR1, CDR2, and CDR3) and/or FR(s) (for example, one or more ofFR1, FR2, FR3, and FR4). The constant region may preferably have a heavychain constant region and/or a light chain constant region, and in termsof the sequence and type, it may be, for example, an IgG-type constantregion (preferably, human IgG1, human IgG2, human IgG3, or humanIgG4-type constant region, human κ chain constant region, and human λchain constant region). It is also possible to use modified variants ofthese constant regions.

In one embodiment, the modification of at least one amino acid residuethat can be exposed on the antibody surface may be either a modificationof a single amino acid or a combination of modifications of multipleamino acids. A preferred method can be to introduce a combination ofmultiple amino acid substitutions at sites where amino acids can beexposed on the antibody surface. Furthermore, without limitations, suchmultiple amino acid substitutions are preferably introduced at positionsthat are three-dimensionally close to one another. When amino acids witha positively charged side chain (for example, Lys (K) or Arg (R)) havebeen substituted for amino acids that can be exposed on the surface ofan antibody molecule (which are preferably, but are not limited to,amino acids with a negatively charged side chain (for example, Glu (E)or Asp (D)); or when pre-existing amino acids having a positively charge(for example, Lys (K) or Arg (R)) are used, for example, one or moreamino acids (which may include amino acids embedded inside the antibodymolecule depending on the situation) that are three-dimensionally closeto the amino acids may also be substituted with amino acids having apositively charge to consequently create a dense state of local positivecharge in a three-dimensionally proximal location. Herein, thedefinition of “a three-dimensionally proximal location” is notparticularly limited; but it can mean a state where one or more aminoacid substitution is introduced, for example, within 20 Å, preferablywithin 15 Å, and more preferably within 10 Å. Whether an amino acidsubstitution site of interest is exposed on the surface of an antibodymolecule or whether an amino acid substitution site is close to otheramino acid substitution site(s) or the above pre-existing amino acidscan be assessed by known methods such as X-ray crystallography.

In addition to those described above, methods for giving multiplepositive charges at sites three-dimensionally close to one another caninclude those that use amino acids that originally have a positivecharge in the native IgG constant region. Such amino acids include, forexample: arginine at positions 255, 292, 301, 344, 355, and 416,according to EU numbering; and lysine at positions 121, 133, 147, 205,210, 213, 214, 218, 222, 246, 248, 274, 288, 290, 317, 320, 322, 326,334, 338, 340, 360, 370, 392, 409, 414, and 439, according to EUnumbering. Multiple positive charges can be given into athree-dimensionally proximal location by substituting with positivelycharged amino acid(s) at sites three-dimensionally close to thesepositively charged amino acids.

Where antibodies of Disclosure A have a variable region (that may bemodified), amino acid residues that are not masked by antigen binding(i.e., that still can be exposed on the surface) may be modified, and/oramino acid modification may not be introduced at sites that are maskedby antigen binding or amino acid modification that does not(substantially) inhibit antigen binding may be carried out. Where aminoacid residues that can be exposed on the surface of an antibody moleculepresent in the ion concentration-dependent binding domain are modified,amino acids of the antigen-binding domain may be modified in such a waythat the modification does not (substantially) reduce the bindingactivity of amino acid residues that can change the antigen-bindingactivity of the antibody according to the ion concentration condition(for example, those in a calcium-binding motif, or a histidine insertionsite and/or a histidine substituted site), or amino acid residues may bemodified at sites other than of the amino acid residues that can changethe antigen-binding activity of the antibody according to the ionconcentration condition. On the other hand, where amino acid residuesthat can be exposed on the surface of an antibody molecule present inthe ion concentration-dependent binding domain have already beenmodified, the type or the position of amino acid residues that canchange the antigen-binding activity of the antibody according to the ionconcentration condition may be selected such that the pI of the antibodyis not reduced below an acceptable level. Where the pI of an antibody isreduced below an acceptable level, the pI of the overall antibody can beincreased by modifying at least one amino acid residue that can beexposed on the surface of the antibody molecule.

Without limitations, FR sequences with a high pI may be preferablyselected from human germline FR sequences or sequences of regions thatare equivalent thereto, whose amino acid may be modified in some cases.

Where antibodies of Disclosure A have a constant region (that may bemodified) having an FcγR-binding domain (which may be a binding domainto any of the FcγR isoforms and allotypes described below) and/or anFcRn-binding domain, sites for modification of at least one amino acidresidue that can be exposed on the surface of the constant region can beamino acid residues other than those in the FcγR-binding domain and/orthose in the FcRn-binding domain, if desired. Alternatively, where themodification sites are selected from amino acid residues in theFcγR-binding domain and/or in the FcRn-binding domain, it may bepreferable to select sites that do not (substantially) affect thebinding activity or binding affinity for FcγR and/or FcRn, or if theywould affect, sites which is biologically or pharmacologicallyacceptable.

In one embodiment, the site of the at least one amino acid residue thatis modified to produce an antibody of Disclosure A whose pI is increasedby modification of at least one amino acid residue that can be exposedon the surface of the variable region (that may be modified) is notlimited; however, such a site can be selected from the group consistingof, according to Kabat numbering: (a) position 1, 3, 5, 8, 10, 12, 13,15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75,76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in a FRof the heavy chain variable region; (b) position 31, 61, 62, 63, 64, 65,and 97 in a CDR of the heavy chain variable region; (c) position 1, 3,7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49,57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103,105, 106, 107, and 108 in a FR of the light chain variable region; and(d) position 24, 25, 26, 27, 52, 53, 54, 55, and 56 in a CDR of thelight chain variable region, wherein an amino acid at each positionafter modification can be selected from any of the amino acids describedabove in terms of the side-chain charge such as Lys (K), Arg (R), Gln(Q), Gly (G), Ser (S), or Asn (N), but is not limited thereto. In someembodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 of theabove amino acid positions are modified. In some embodiments, 1-20,1-15, 1-10, or 1-5 of the above amino acid positions are modified.

In one embodiment, among the position(s) to be modified, the followingposition(s) can be used for aiding in pI increase of an antibody ofDisclosure A, in combination with other position(s) which themselves canhave sufficient effect of increasing pI of an antibody. Such position(s)for aiding in the pI increase can be, for example, as for a light chainvariable region, selected from a group consisting of positions 27, 52,56, 65, and 69, according to Kabat numbering.

Furthermore, the site of at least one amino acid residue that ismodified in the CDR and/or FR is not limited; however, such a site canbe selected from the group consisting of: (a) position 8, 10, 12, 13,15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85, and 105 inthe FR of the heavy chain variable region; (b) position 31, 61, 62, 63,64, 65, and 97 in the CDR of the heavy chain variable region; (c)position 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107 in theFR of the light chain variable region; and (d) position 24, 25, 26, 27,52, 53, 54, 55, and 56 in the CDR of the light chain variable region. Insome embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 ofthe above amino acid positions are modified. In some embodiments, 1-20,1-15, 1-10, or 1-5 of the above amino acid positions are modified.

Where the modification site of at least one amino acid residue isselected, for example, from a group comprising the above-describedgroups, the type of amino acid after modification in the heavy-chainvariable region is, for example:

(a) 8K, 8R, 8Q, 8G, 8S, or 8N for position 8; (b) 13K, 13R, 13Q, 13G,13S, or 13N for position 13; (c) 15K, 15R, 15Q, 15G, 15S, or 15N forposition 15; (d) 16K, 16R, 16Q, 16G, 16S, or 16N for position 16; (e)18K, 18R, 18Q, 18G, 18S, or 18N for position 18; (f) 39K, 39R, 39Q, 39G,39S, or 39N for position 39; (g) 41K, 41R, 41Q, 41G, 41S, or 41N forposition 41; (h) 43K, 43R, 43Q, 43G, 43S, or 43N for position 43; (i)44K, 44R, 44Q, 44G, 44S, or 44N for position 44; (j) 63K, 63R, 63Q, 63G,63S, or 63N for position 63; (k) 64K, 64R, 64Q, 64G, 64S, or 64N forposition 64; (l) 77K, 77R, 77Q, 77G, 77S, or 77N for position 77; (m)82K, 82R, 82Q, 82G, 82S, or 82N for position 82; (n) 82aK, 82aR, 82aQ,82aG, 82aS, or 82aN for position 82a; (o) 82bK, 82bR, 82bQ, 82bG, 82bS,or 82bN for position 82b; (p) 83K, 83R, 83Q, 83G, 83S, or 83N forposition 83; (q) 84K, 84R, 84Q, 84G, 84S, or 84N for position 84; (r)85K, 85R, 85Q, 85G, 85S, or 85N for position 85; or (s) 105K, 105R,105Q, 105G, 105S, or 105N for position 105. In some embodiments, atleast 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 of any combination ofthe above amino acid positions are modified. In some embodiments, 1-20,1-15, 1-10, or 1-5 of any combination of the above amino acid positionsare modified.

Non-limiting examples of a combination of modified amino acids positionsin the heavy-chain variable region is, for example:

any two or more of positions selected from the group consisting ofpositions 16, 43, 64, and 105; any two or more of positions selectedfrom the group consisting of positions 77, 82a, and 82b; positions 77and 85; positions 41 and 44; positions 82a and 82b; positions 82 and82b; positions 82b and 83; or positions 63 and 64, according to Kabatnumbering, wherein an amino acid at each position after modification canbe selected from any of the amino acids described above in terms of theside-chain charge such as Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S),or Asn (N), but is not limited thereto.

A specific combination can be, for example, 16Q/43R/64K/105Q;77R/82aN/82bR; 77R/82aG/82bR; 77R/82aS/82bR; 77R/85G; 41R/44R;82aN/82bR; 82aG/82bR; 82aS/82bR; 82K/82bR; 82bR/83R; 77R/85R; or63R/64K.

Likewise, the type of amino acid after modification in the light-chainvariable region is, for example: (a) 16K, 16R, 16Q, 16G, 16S, or 16N forposition 16; (b) 18K, 18R, 18Q, 18G, 18S, or 18N for position 18; (c)24K, 24R, 24Q, 24G, 24S, or 24N for position 24; (d) 25K, 25R, 25Q, 25G,25S, or 25N for position 25; (e) 26K, 26R, 26Q, 26G, 26S, or 26N forposition 26; (f) 27K, 27R, 27Q, 27G, 27S, or 27N for position 27; (g)37K, 37R, 37Q, 37G, 37S, or 37N for position 37; (h) 41K, 41R, 41Q, 41G,41S, or 41N for position 41; (i) 42K, 42R, 42Q, 42G, 42S, or 42N forposition 42; (j) 45K, 45R, 45Q, 45G, 45S, or 45N for position 45; (k)52K, 52R, 52Q, 52G, 52S, or 52N for position 52; (l) 53K, 53R, 53Q, 53G,53S, or 53N for position 53; (m) 54K, 54R, 54Q, 54G, 54S, or 54N forposition 54; (n) 55K, 55R, 55Q, 55G, 55S, or 55N for position 55; (o)56K, 56R, 56Q, 56G, 56S, or 56N for position 56; (p) 65K, 65R, 65Q, 65G,65S, or 65N for position 65; (q) 69K, 69R, 69Q, 69G, 69S, or 69N forposition 69; (r) 74K, 74R, 74Q, 74G, 74S, or 74N for position 74; (s)76K, 76R, 76Q, 76G, 76S, or 76N for position 76; (t) 77K, 77R, 77Q, 77G,77S, or 77N for position 77; (u) 79K, 79R, 79Q, 79G, 79S, or 79N forposition 79; and (v) 107K, 107R, 107Q, 107G, 107S, or 107N for position107. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or morethan 10 of any combination of the above amino acid positions aremodified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of anycombination of the above amino acid positions are modified.

Non-limiting examples of a combination of modified amino acids positionsin the light-chain variable region is, for example: positions 24 and 27;positions 25 and 26; positions 41 and 42; positions 42 and 76; positions52 and 56; positions 65 and 79; positions 74 and 77; positions 76 and79; any two or more of positions selected from the group consisting ofpositions 16, 24, and 27; any two or more of positions selected from thegroup consisting of positions 24, 27, and 37; any two or more ofpositions selected from the group consisting of positions 25, 26, and37; any two or more of positions selected from the group consisting ofpositions 27, 76, and 79; any two or more of positions selected from thegroup consisting of positions 41, 74, and 77; any two or more ofpositions selected from the group consisting of positions 41, 76, and79; any two or more of positions selected from the group consisting ofpositions 24, 27, 41, and 42; any two or more of positions selected fromthe group consisting of positions 24, 27, 52, and 56; any two or more ofpositions selected from the group consisting of positions 24, 27, 65,and 69; any two or more of positions selected from the group consistingof positions 24, 27, 74, and 77; any two or more of positions selectedfrom the group consisting of positions 24, 27, 76, and 79; any two ormore of positions selected from the group consisting of positions 25,26, 52, and 56; any two or more of positions selected from the groupconsisting of positions 25, 26, 65, and 69; any two or more of positionsselected from the group consisting of positions 25, 26, 76, and 79; anytwo or more of positions selected from the group consisting of positions27, 41, 74, and 77; any two or more of positions selected from the groupconsisting of positions 27, 41, 76, and 79; any two or more of positionsselected from the group consisting of positions 52, 56, 74, and 77; anytwo or more of positions selected from the group consisting of positions52, 56, 76, and 79; any two or more of positions selected from the groupconsisting of positions 65, 69, 76, and 79; any two or more of positionsselected from the group consisting of positions 65, 69, 74, and 77; anytwo or more of positions selected from the group consisting of positions18, 24, 45, 79, and 107; any two or more of positions selected from thegroup consisting of positions 27, 52, 56, 74, and 77; any two or more ofpositions selected from the group consisting of positions 27, 52, 56,76, and 79; any two or more of positions selected from the groupconsisting of positions 27, 65, 69, 74, and 77; any two or more ofpositions selected from the group consisting of positions 27, 65, 69,76, and 79; any two or more of positions selected from the groupconsisting of positions 41, 52, 56, 74, and 77; any two or more ofpositions selected from the group consisting of positions 41, 52, 56,76, and 79; any two or more of positions selected from the groupconsisting of positions 41, 65, 69, 74, and 77; any two or more ofpositions selected from the group consisting of positions 41, 65, 69,76, and 79; any two or more of positions selected from the groupconsisting of positions 24, 27, 41, 42, 65, and 69; any two or more ofpositions selected from the group consisting of positions 24, 27, 52,56, 65, and 69; any two or more of positions selected from the groupconsisting of positions 24, 27, 65, 69, 74, and 77; any two or more ofpositions selected from the group consisting of positions 24, 27, 65,69, 76, and 79; any two or more of positions selected from the groupconsisting of positions 24, 27, 41, 42, 74, and 77; any two or more ofpositions selected from the group consisting of positions 24, 27, 52,56, 74, and 77; any two or more of positions selected from the groupconsisting of positions 24, 27, 41, 42, 76, and 79; any two or more ofpositions selected from the group consisting of positions 24, 27, 52,56, 76, and 79; any two or more of positions selected from the groupconsisting of positions 24, 27, 74, 76, 77, and 79; any two or more ofpositions selected from the group consisting of positions 52, 56, 65,69, 74, and 77; or any two or more of positions selected from the groupconsisting of positions 52, 56, 65, 69, 76, and 79, according to Kabatnumbering, wherein an amino acid at each position after modification canbe selected from any of the amino acids described above in terms of theside-chain charge such as Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S),or Asn (N), but is not limited thereto.

A specific combination can be, for example, 24R/27Q; 24R/27R; 24K/27K;25R/26R; 25K/26K; 41R/42K; 42K/76R; 52R/56R; 65R/79K; 74K/77R; 76R/79K;16K/24R/27R; 24R/27R/37R; 25R/26R/37R; 27R/76R/79K; 41R/74K/77R;41R/76R/79K; 24R/27R/41R/42K; 24R/27R/52R/56R; 24R/27R/52K/56K;24R/27R/65R/69R; 24R/27R/74K/77R; 24R/27R/76R/79K; 25R/26R/52R/56R;25R/26R/52K/56K; 25R/26R/65R/69R; 25R/26R/76R/79K; 27R/41R/74K/77R;27R/41R/76R/79K; 52R/56R/74K/77R; 52R/56R/76R/79K; 65R/69R/76R/79K;65R/69R/74K/77R; 18R/24R/45K/79Q/107K; 27R/52R/56R/74K/77R;27R/52R/56R/76R/79K; 27R/65R/69R/74K/77R; 27R/65R/69R/76R/79K;41R/52R/56R/74K/77R; 41R/52R/56R/76R/79K; 41R/65R/69R/74K/77R;41R/65R/69R/76R/79K; 24R/27R/41R/42K/65R/69R; 24R/27R/52R/56R/65R/69R;24R/27R/65R/69R/74K/77R; 24R/27R/65R/69R/76R/79K;24R/27R/41R/42K/74K/77R; 24R/27R/52R/56R/74K/77R;24R/27R/41R/42K/76R/79K; 24R/27R/52R/56R/76R/79K;24R/27R/74K/76R/77R/79K; 52R/56R/65R/69R/74K/77R; or52R/56R/65R/69R/76R/79K.

In WO2007/114319 or WO2009/041643, it has already been explained ordemonstrated based on theoretical evidence, homology modeling, orexperimental techniques that the effect of increasing the pI viamodification of some amino acid residues in the variable region does notexclusively (or substantially) depend on the antibody-constituting aminoacid sequences per se or the type of target antigen, but rather itdepends on the type and number of amino acid residues that aresubstituted. It has been also demonstrated that even after modificationof some amino acids, the antigen-binding activity for several types ofantigens is (substantially) maintained, or at least can be expected tobe maintained with high possibility by those of ordinary skill in theart.

For example, WO2009/041643 specifically shows that in the heavy-chain FRof a humanized glypican 3 antibody as shown in SEQ ID NO:8, preferredmodification sites of amino acid residues that can be exposed on thesurface are positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26,39, 42, 43, 44, 46, 69, 72, 73, 74, 76, 77, 82, 85, 87, 89, 90, 107,110, 112, and 114 according to Kabat numbering. It also reports that theamino acid residue at position 97 according to Kabat numbering ispreferred because it is exposed on the surface of almost all antibodies.WO2009/041643 also shows that the amino acid residues of positions 52,54, 62, 63, 65, and 66 in the heavy-chain CDR of the antibody arepreferred. It also shows that the amino acid residues of positions 1, 3,7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 43, 44, 45, 46, 48, 49, 50, 54, 62,65, 68, 70, 71, 73, 74, 75, 79, 81, 82, 84, 85, 86, 90, 105, 108, 110,111, and 112 according to Kabat numbering in the light-chain FR of ahumanized glypican 3 antibody as shown in SEQ ID NO:9 are preferred. Italso shows that the amino acid residues of positions 24, 27, 33, 55, 59in the light-chain CDR of this antibody are preferred. Furthermore,WO2009/041643 specifically shows that the amino acid residues ofpositions 31, 64, and 65 according to Kabat numbering in the heavy-chainCDR of an anti-human IL-6 receptor antibody as shown in SEQ ID NO:10 arepreferred sites that allow modification of amino acid residues that canbe exposed on the surface while maintaining the antigen-bindingactivity. It also shows that the amino acid residues of positions 24,27, 53, and 55 according to Kabat numbering in the light chain CDR of ananti-human IL-6 receptor antibody as shown in SEQ ID NO:11 arepreferred. It also specifically shows that the amino acid residue ofposition 31 according to Kabat numbering in the heavy-chain CDR of ananti-human IL-6 receptor antibody as shown in SEQ ID NO:12 is apreferred site that allows modification of amino acid residue that canbe exposed on the surface while maintaining the antigen-bindingactivity. It also shows that the amino acid residues of positions 24,53, 54, and 55 according to Kabat numbering in the light-chain CDR of ananti-human IL-6 receptor antibody as shown in SEQ ID NO:13 arepreferred. WO2009/041643 also shows that the amino acid residues ofpositions 61, 62, 64, and 65 according to Kabat numbering in theheavy-chain CDR of an anti-human glypican 3 antibody as shown in SEQ IDNO:14 are preferred sites that allow modification of amino acid residuesthat can be exposed on the surface while maintaining the antigen-bindingactivity. It also shows that the amino acid residues of positions 24 and27 according to Kabat numbering in the light-chain CDR of an anti-humanglypican 3 antibody as shown in SEQ ID NO:15 are preferred. It alsoshows that the amino acid residues of positions 61, 62, 64, and 65according to Kabat numbering in the heavy-chain CDR of an anti-humanIL-31 receptor antibody as shown in SEQ ID NO:16 are preferred sitesthat allow modification of amino acid residues that can be exposed onthe surface while maintaining the antigen-binding activity.WO2009/041643 also shows that the amino acid residues of positions 24and 54 according to Kabat numbering in the light-chain CDR of ananti-human IL-31 receptor antibody as shown in SEQ ID NO:17 arepreferred. Similarly, WO2007/114319 reports that antibodies hA69-PF,hA69-p18, hA69-N97R, hB26-F123e4, hB26-p15, and hB26-PF, which wereproduced by modifying the charge of one or more amino acid residues thatcan be exposed on the surface, showed changes in pI as demonstrated byisoelectric focusing, and had an equivalent binding activity to FactorIXa or Factor X, which are their antigens, compared with that of theantibodies before modification or alteration. It also reports that whenthese antibodies were administered to mice, the pI of each antibodyshowed high correlation with their clearance (CL) in plasma, retentiontime in plasma, and half-life in plasma (T1/2). WO2007/114319 alsodemonstrates that amino acid residues of positions 10, 12, 23, 39, 43,97, and 105 in the variable region are preferred as sites formodification of amino acid residues that can be exposed on the surface.

In an alternative or further embodiment, for example, using knownmethods such as X-ray crystallography or a homology model constructed byhomology modeling from an antibody constant region (which is preferablya human constant region, more preferably a human Ig-type constantregion, and still more preferably a human IgG-type constant region, butis not limited thereto), amino acid residues that can be exposed on thesurface of an antibody constant region may be identified to determinethe modification sites of at least one amino acid residue for producingan antibody of Disclosure A whose pI has been increased. Themodification site of at least one amino acid residue that can be exposedon the surface of the constant region is not limited; however, the sitecan be preferably selected from the group consisting of: position 196,253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308,309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373,382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415, 418,419, 421, 424, 430, 433, 434, and position 443, according to EUnumbering, and may be preferably selected from the group consisting of:position 254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 342, 343,345, 356, 358, 359, 361, 362, 384, 385, 386, 387, 389, 399, 400, 401,402, 413, 418, 419, 421, 433, 434, and 443, and may be also preferablyselected from the group consisting of: positions 282, 309, 311, 315,342, 343, 384, 399, 401, 402, and 413, whose amino acid at each positionafter modification can be selected from the amino acids described abovein terms of the side-chain charge such as Lys (K), Arg (R), Gln (Q), orAsn (N), but is not limited thereto. When the modification site of atleast one amino acid residue is selected, for example, from a groupcomprising the above-described groups, for example, the type of aminoacid after modification at each site can be as follows:

254K, 254R, 254Q, or 254N at position 254; 258K, 258R, 258Q, or 258N atposition 258; 281K, 281R, 281Q, or 281N at position 281; 282K, 282R,282Q, or 282N at position 282; 285K, 285R, 285Q, or 285N at position285; 309K, 309R, 309Q, or 309N at position 309; 311K, 311R, 311Q, or311N at position 311; 315K, 315R, 315Q, or 315N at position 315; 327K,327R, 327Q, or 327N at position 327; 330K, 330R, 330Q, or 330N atposition 330; 342K, 342R, 342Q, or 342N at position 342; 343K, 343R,343Q, or 343N at position 311; 345K, 345R, 345Q, or 345N at position345; 356K, 356R, 356Q, or 356N at position 356; 358K, 358R, 358Q, or358N at position 358; 359K, 359R, 359Q, or 359N at position 359; 361K,361R, 361Q, or 361N at position 361; 362K, 362R, 362Q, or 362N atposition 362; 384K, 384R, 384Q, or 384N at position 384; 385K, 385R,385Q, or 385N at position 385; 386K, 386R, 386Q, or 386N at position386; 387K, 387R, 387Q, or 387N at position 387; 389K, 389R, 389Q, or389N at position 389; 399K, 399R, 399Q, or 399N at position 399; 400K,400R, 400Q, or 400N at position 400; 401K, 401R, 401Q, or 401N atposition 401; 402K, 402R, 402Q, or 402N at position 402; 413K, 413R,413Q, or 413N at position 413; 418K, 418R, 418Q, or 418N at position418; 419K, 419R, 419Q, or 419N at position 419; 421K, 421R, 421Q, or421N at position 421; 433K, 433R, 433Q, or 433N at position 433; 434K,434R, 434Q, or 434N at position 434; and 443K, 443R, 443Q, or 443N atposition 443.

In an alternative embodiment, the modification site of at least oneamino acid residue and the type of amino acid after modification mayinclude 345R or 345K, and/or 430R, 430K, 430G, or 435T, according to EUnumbering.

In one embodiment of the antibodies of Disclosure A, the antibody's netpI may be increased by modifying at least one amino acid residue thatcan be exposed on the surface of the variable region (which may bemodified) as described above and at least one amino acid residue thatcan be exposed on the surface of the constant region (which may bemodified) as described above.

Within the scope of Disclosures A and B described herein, where anantibody of Disclosure A or B is an IgG-type antibody or a moleculederived therefrom, the antibody heavy-chain constant region may containa constant region of the IgG1 type, IgG2 type, IgG3 type, or IgG4 type.In Disclosure A or B, the heavy-chain constant region may be a humanheavy-chain constant region, but is not limited thereto. Severalallotypes are known to exist for human IgG. Specifically, it has beenreported that there are some differences in the amino acid sequence ofthe human IgG constant region among individuals (Methods Mol. Biol.882:635-80 (2012); Sequences of proteins of immunological interest, NIHPublication No. 91-3242). Examples include human IgG1 constant region(SEQ ID NO:18), human IgG2 constant region (SEQ ID NO:19), human IgG3constant region (SEQ ID NO:20), and human IgG4 constant region (SEQ IDNO:21).

Of these, for example, allotypes called G1m1,17 and G1m3 are known forhuman IgG1. The allotypes differ in their amino acid sequences: G1m1,17has aspartic acid at position 356 and leucine at position 358 accordingto EU numbering, while G1m3 has glutamic acid at position 356 andmethionine at position 358 according to EU numbering. There is, however,no report suggesting the presence of significant differences inessential antibody functions and properties among the reportedallotypes. Thus, those of ordinary skill in the art can readily predictthat various assessments were performed using specific allotypes, andthe results are not limited to the allotypes used to obtain the Examplesand the same effects are expected with any allotypes. Within the scopeof Disclosures A and B described herein, when noted as “human IgG1”,“human IgG2, “human IgG3”, or “human IgG4”, the allotypes are notlimited to specific allotypes and can include all reported allotypes.

In an alternative or further embodiment of Disclosure A or B, thelight-chain constant region of an antibody can include any constantregion of the κ chain (IgK) type or λ chain (IgL1, IgL2, IgL3, IgL6, orIgL7) type. A light-chain constant region may be preferably a humanlight-chain constant region, but is not limited thereto. There arereports, such as in Sequences of proteins of immunological interest, NIHPublication No. 91-3242, on several allotype sequences that result fromgene polymorphism for the human κ chain constant region and human λchain constant region. Such allotypes include, for example, human κchain constant region (SEQ ID NO:22) and human λ chain constant region(SEQ ID NO:23). There is, however, no report suggesting the presence ofsignificant differences in essential antibody functions and propertiesamong the reported allotypes. Thus, those of ordinary skill in the artcan readily understand that when reference is made to specific allotypeswithin the scope of Disclosures A and B described herein, the sameeffects are expected with any allotypes (hereinafter, also collectivelyreferred to as native (human) IgG (type) constant region).

Moreover, since the Fc region of a native IgG antibody constitutes apart of the constant region of the native IgG antibody, when antibodiesof Disclosure A or B are, for example, IgG type antibodies or moleculesderived therefrom, the antibodies may have an Fc region contained in theconstant region of a native IgG (IgG1, IgG2, IgG3, or IgG4 type)(hereinafter, also collectively referred to as a native (human) IgG(type) Fc region). The Fc region of a native IgG can refer to an Fcregion consisting of the same amino acid sequence as an Fc regionoriginating from a naturally occurring IgG. Specific examples of the Fcregion of a native human IgG can include the Fc regions contained in thehuman IgG1 constant region (SEQ ID NO:18), human IgG2 constant region(SEQ ID NO:19), human IgG3 constant region (SEQ ID NO:20), or human IgG4constant region (SEQ ID NO:21) described above (an Fc region of the IgGclass can refer to, for example, from cysteine of position 226 accordingto EU numbering to the C terminus, or from proline of position 230according to EU numbering to the C terminus.).

In one embodiment, antibodies of Disclosures A and B may includevariants in which one or more modifications selected from amino acidsubstitution, addition, deletion, or insertion have been made to theconstant region of a native (preferably human) IgG (the heavy-chainconstant region and/or the light-chain constant region) or in the Fcregion of a native (preferably human) IgG.

Within the scope of Disclosure A described herein, WO2013/081143 reportsthat for example, ion concentration-dependent antibodies capable offorming multivalent immune complexes with a multimeric antigen(multivalent antigen-antibody complexes) and multispecific ionconcentration-dependent antibodies or multiparatopic ionconcentration-dependent antibodies that can form multivalent immunecomplexes (multivalent antigen-antibody complexes) by recognizing two ormore epitopes on monomeric antigens can bind more strongly to FcγR,FcRn, complement receptor, due to the avidity (sum of the strength ofbinding between multiple epitopes and multiple paratopes) via at leasttwo or more multivalent constant regions (that may be modified) or Fcregions (that may be modified) contained in the antibody molecules, andas a result the antibodies are more rapidly taken up into cells. Thus,when modified to have an increased pI via modification of at least oneamino acid residue that can be exposed on the antibody surface, the ionconcentration-dependent antibodies described above, which are capable offorming multivalent immune complexes with a multimeric antigen ormonomeric antigens, can also be used as antibodies of Disclosure A (ionconcentration-dependent antibodies with increased pI). Those of ordinaryskill in the art will appreciate that the ion concentration-dependentantibodies with increased pI that can form multivalent immune complexeswith a multimeric antigen or monomeric antigens can be more rapidlytaken up into cells, as compared to ion concentration-dependentantibodies with increased pI that are incapable of forming multivalentimmune complexes. Those of ordinary skill in the art can also understandthat in one embodiment, the activity of antibodies of Disclosure A tobind to FcRn and/or FcγR may be increased under a neutral pH conditionand in this case, the ion concentration-dependent antibodies withincreased pI that can form multivalent immune complexes with amultimeric antigen or monomeric antigens may be even more rapidly takenup into cells.

In one embodiment, antibodies of Disclosure A may be one-armedantibodies (including all embodiments of the one-armed antibodiesdescribed in WO2005/063816). Typically, one-armed antibodies areantibodies that lack one of the two Fab regions an ordinary IgG antibodyhas, and can be produced, without limitations, for example, by themethods described in WO2005/063816. Without limitations, in an IgG-typeantibody that has a heavy chain whose structure is, for example,VH-CH1-Hinge-CH2-CH3, when one of the Fab regions is cleaved at a sitemore to the N terminus than the Hinge (for example, VH or CH1), theantibody will be expressed in a form containing an extra sequence, andwhen one of the Fab regions is cleaved at a site more to the C terminusthan the Hinge (for example, CH2), the Fc region will have an incompleteform. Thus, without limitations, it is preferable from the viewpoint ofantibody molecule stability that one-armed antibodies are produced bycleavage in the hinge region (Hinge) of one of the two Fab regions of anIgG antibody. It is more preferable that the heavy chain after cleavageis linked to the uncleaved heavy chain via intramolecular disulfidebond. WO2005/063816 has reported that such one-armed antibodies have anincreased stability as compared to Fab molecules. Antibodies with anincreased or decreased pI can also be generated by preparing suchone-armed antibodies. Furthermore, when an ion concentration-dependentantigen-binding domain is introduced into antibodies with an increasedpI that are one-armed antibodies, the antibody half-life in plasma canbe further shortened, cellular uptake of the antibody can be furtherenhanced, antigen elimination from plasma can be further enhanced, orthe antibody's affinity for the extracellular matrix can be furtherincreased, as compared to antibodies with increased pI that do not havean ion concentration-dependent antigen-binding domain.

Without being bound by a particular theory, an embodiment where thecellular uptake-accelerating effect of one-armed antibodies is expectedcan be envisaged to be, but is not limited to, a case in which the pI ofa soluble antigen is lower than that of the antibodies. The net pI of acomplex consisting of antibodies and antigens can be calculated by knownmethods by considering that the complex is a single molecule. In thiscase, the lower the pI of the soluble antigen is, the lower the net pIof the complex is; and the greater the pI of the soluble antigen is, thegreater the net pI of the complex is. When an ordinary-type IgG antibodymolecule (having two Fabs) is bound to a single low-pI soluble antigenversus to two low-pI soluble antigens, the net pI of the complex islower in the latter case. When such an ordinary-type antibody isconverted into a one-armed antibody, only one antigen can bind to asingle molecule of the antibody; reduction of the pI of the complexresulting from the binding of the second antigen can thereby besuppressed. In other words, it is believed that when the pI of thesoluble antigen is lower than that of the antibody, the conversion intoa one-armed antibody allows the pI of the complex to increase ascompared to an ordinary antibody, and thereby accelerates uptake intocells.

Furthermore, without limitations, when the Fab of an ordinary IgG-typeantibody molecule (having two Fabs) has a lower pI than that of the Fc,conversion into a one-armed antibody increases the net pI of the complexconsisting of the one-armed antibody and antigen. Moreover, when suchconversion into a one-armed antibody is performed, it is preferable fromthe viewpoint of the stability of the one-armed antibody that one of theFabs is cleaved in the Hinge region located at the junction between Faband Fc. In this case, the pI can be expected to be effectively increasedby selecting a site which would increase the pI of the one-armedantibody to the desired extent.

Thus, those of ordinary skill in the art can understand that withoutexclusively (or substantially) depending on the antibody amino acidsequence itself and the type of the soluble antigen, the pI of anantibody can be increased and the accompanying cellular uptake of theantigen may be accelerated by converting the antibody into a one-armedantibody by calculating the theoretical pI of the antibody (theoreticalpI of Fc and theoretical pI of Fab) and the theoretical pI of thesoluble antigen and predicting the relationship on the difference oftheir theoretical pI values.

In one embodiment, antibodies of Disclosure A or B may be multispecificantibodies, and the multispecific antibody may be, but is not limitedto, a bispecific antibody. The multispecific antibody may be amultispecific antibody that contains a first polypeptide and a secondpolypeptide. Here, “a multispecific antibody that contains a firstpolypeptide and a second polypeptide” refers to an antibody that bindsto at least two or more types of different antigens or at least two ormore types of epitopes in a same antigen. The first polypeptide andsecond polypeptide preferably may contain a heavy-chain variable region,and more preferably the variable region contains CDR(s) and/or FR(s). Inanother embodiment, the first polypeptide and second polypeptide maypreferably each contain a heavy-chain constant region. In still anotherembodiment, the multispecific antibody may contain a third polypeptideand a fourth polypeptide, each containing a light-chain variable regionand preferably also a light-chain constant region. In this case, thefirst to the fourth polypeptides may assemble together to form amultispecific antibody.

In one embodiment, where antibodies of Disclosure A are multispecificantibodies and the multispecific antibodies contain a heavy-chainconstant region, to reduce their pI, for example, the followingsequences may be used: IgG2 or IgG4 sequence at position 137; IgG1,IgG2, or IgG4 sequence at position 196; IgG2 or IgG4 sequence atposition 203; IgG2 sequence at position 214; IgG1, IgG3, or IgG4sequence at position 217; IgG1, IgG3, or IgG4 sequence at position 233;IgG4 sequence at position 268; IgG2, IgG3, or IgG4 sequence at position274; IgG1, IgG2, or IgG4 sequence at position 276; IgG4 sequence atposition 355; IgG3 sequence at position 392; IgG4 sequence at position419; or IgG1, IgG2, or IgG4 sequence at position 435. Meanwhile, toincrease their pI, for example, the following sequences may be used:IgG1 or IgG3 sequence at position 137; IgG3 sequence at position 196;IgG1 or IgG3 sequence at position 203; IgG1, IgG3, or IgG4 sequence atposition 214; IgG2 sequence at position 217; IgG2 sequence at position233; IgG1, IgG2, or IgG3 sequence at position 268; IgG1 sequence atposition 274; IgG3 sequence at position 276; IgG1, IgG2, or IgG3sequence at position 355; IgG1, IgG2, or IgG4 sequence at position 392;IgG1, IgG2, or IgG3 sequence at position 419; or IgG3 sequence atposition 435.

In one embodiment, where antibodies of Disclosure A have two heavy-chainconstant regions, the pIs of the two heavy chain constant regions may bethe same or different from each other. Such heavy-chain constant regionsmay be IgG1, IgG2, IgG3 and IgG4 heavy-chain constant regions whichoriginally have different pIs. Alternatively, it is possible tointroduce a pI difference between the two heavy-chain constant regions.Modification sites of at least one amino acid residue for introducingsuch a pI difference in the constant region may be the position(s)described above or position(s) selected, for example, from the groupconsisting of position 137, position 196, position 203, position 214,position 217, position 233, position 268, position 274, position 276,position 297, position 355, position 392, position 419, and position435, according to EU numbering in the heavy-chain constant region asdescribed in WO2009/041643. Alternatively, the amino acid residue ofposition 297 which is a glycosylation site may be modified to remove thesugar chain, since the removal of a sugar chain from the heavy-chainconstant region results in a pI difference.

In one embodiment, antibodies of Disclosure A or B may be polyclonalantibodies or monoclonal antibodies, and mammalian-derived monoclonalantibodies are preferred. Monoclonal antibodies include those producedby hybridomas or those produced by host cells transformed by geneticengineering techniques with expression vectors carrying antibody genes.The antibodies of Disclosure A or B may be, for example, antibodies suchas chimeric antibodies, humanized antibodies, or antibodies generated byaffinity maturation, or molecules derived therefrom.

In one embodiment, antibodies of Disclosure A or B may be derived,without limitations, from any animal species (for example, human; ornonhuman animals such as mouse, rat, hamster, rabbit, monkey, cynomolgusmonkey, Rhesus monkey, hamadryas baboon, chimpanzee, goat, sheep, dog,bovine, or camel), or any birds; and the antibodies are preferablyderived from human, monkey, or mouse.

In one embodiment, antibodies of Disclosure A or B may be Ig-typeantibodies, and may be preferably IgG-type antibodies.

Within the scope of Disclosures A and B described herein, the Fcreceptor (also referred to as “FcR”) refers to a receptor protein thatcan bind to the Fc region of an immunoglobulin (antibody) or a moleculederived therefrom, or an Fc region variant. For example, Fc receptorsfor IgG, IgA, IgE, and IgM are known as FcγR, FcαR, FcεR, and FcμR,respectively, within the scope of Disclosure A described herein. Fcreceptors may also be, for example, FcRn (also referred to as “neonatalFc receptor”), within the scope of Disclosures A and B described herein.

Within the scope of Disclosure A described herein, “FcγR” may refer to areceptor protein that can bind to the Fc region of an IgG1, IgG2, IgG3,or IgG4 antibody or a molecule derived therefrom, or an Fc regionvariant, and may include any one or more of, or all members of thefamily of proteins substantially encoded by the FcγR gene. In human, thefamily includes, but is not limited to, FcγRI (CD64) including isoformsFcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoforms FcγRIIa(including allotypes H131 (type H) and R131 (type R)), FcγRIIb(including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16)including isoforms FcγRIIIa (including allotypes V158 and F158) andFcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well asall unidentified human FcγRs and FcγR isoforms and allotypes.Furthermore, FcγRIIb1 and FcγRIIb2 have been reported as splicingvariants of human FcγRIIb (hFcγRIIb). There is also a report on asplicing variant called FcγRIIb3 (Brooks et al., J. Exp. Med, 170:1369-1385 (1989)). In addition to those described above, hFcγRIIbincludes all splicing variants such as those registered in NCBI underNP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, andNP_003992.3. hFcγRIIb also includes all genetic polymorphisms alreadyreported, for example, FcγRIIb (Li et al., Arthritis Rheum. 48:3242-3252(2003), Kono et al., Hum. Mol. Genet. 14:2881-2892 (2005); Kyogoku etal., Arthritis Rheum. 46(5):1242-1254 (2002)), as well as all geneticpolymorphisms that will be reported in future.

FcγR may be derived from any organism, and may include those derivedfrom humans, mice, rats, rabbits, or monkeys, without being limitedthereto. Mouse FcγRs include, but are not limited to, FcγRI (CD64),FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), as well as allunidentified mouse FcγRs, and FcγR isoforms and allotypes. Suchpreferred FcγR includes, for example, human FcγRI (CD64), FcγRIIA(CD32), FcγRIIB (CD32), FcγRIIIA (CD16), or FcγRIIIB (CD16). Since FcγRis present as a membrane form in vivo, it may be used in experimentalsystems after being artificially converted into an appropriate solubleform.

For example, as shown in WO2014/163101, the polynucleotide sequence andamino acid sequence of FcγRI may be the sequences shown in NM 000566.3and NP_000557.1, respectively; the polynucleotide sequence and aminoacid sequence of FcγRIIA may be the sequences shown in BC020823.1 andAAH20823.1, respectively; the polynucleotide sequence and amino acidsequence of FcγRIIB may be the sequences shown in BC146678.1 andAAI46679.1, respectively; the polynucleotide sequence and amino acidsequence of FcγRIIIA may be the sequences shown in BC033678.1 andAAH33678.1, respectively; the polynucleotide sequence and amino acidsequence of FcγRIIIB may be the sequences shown in BC128562.1 andAAI28563.1, respectively (RefSeq accession numbers are shown).

FcγRIIa has two genetic polymorphisms, in which the amino acid atposition 131 of FcγRIIa is replaced with histidine (type H) or arginine(type R) (J. Exp. Med. 172:19-25, 1990).

In FcγRI (CD64) which includes FcγRIa, FcγRIb, and FcγRIc, and FcγRIII(CD16) which includes FcγRIIIa (including allotypes V158 and F158), theα chain that binds to the Fc region of IgG is associated with a common γchain having ITAM which transmits activation signals inside cells.FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) is a GPIanchor protein. Meanwhile, the cytoplasmic domain of FcγRII (CD32) whichincludes the FcγRIIa (including allotypes H131 and R131) and FcγRIIcisoforms contains ITAM. These receptors are expressed on many immunecells such as macrophages, mast cells, and antigen-presenting cells. Theactivation signals transduced upon binding of these receptors to the Fcregion of IgG promote the phagocytotic ability of macrophages,production of inflammatory cytokines, degranulation of mast cells, andthe increased function of antigen-presenting cells. An FcγR that has theability to transduce activation signals as described above is alsoreferred to as an activating FcγR within the scope of Disclosures A andB described here.

Meanwhile, the cytoplasmic domain of FcγRIIb (including FcγRIIb-1 andFcγRIIb-2) contains ITIM which transmits inhibitory signals. In B cells,the crosslinking between FcγRIIb and B cell receptor (BCR) suppressesthe activation signals from BCR, which results in suppression ofantibody production by BCR. In macrophages, the crosslinking of FcγRIIIand FcγRIIb suppresses the phagocytic ability and the ability to produceinflammatory cytokines. An FcγR that has the ability to transduceinhibitory signals as described above is also referred to as aninhibitory Fcγreceptor within the scope of Disclosures A and B describedherein.

Within the scope of Disclosure A described herein, whether the bindingactivity of an antibody or Fc region (variant) toward various FcγRs hasbeen increased, (substantially) maintained, or reduced as compared tothe antibody or Fc region (variant) before modification can be assessedby methods known to those of ordinary skill in the art. Such methods arenot particularly limited and those described in the present Examples maybe used, and for example, surface plasmon resonance (SPR)phenomenon-based BIACORE (Proc. Natl. Acad. Sci. USA (2006) 103(11),4005-4010) may be used. Alternatively, for example, ELISA andfluorescence activated cell sorting (FACS) as well as ALPHA screen(Amplified Luminescent Proximity Homogeneous Assay) may be used. Inthese assays, the extracellular domain of human FcγR may be used as asoluble antigen (for example, WO2013/047752).

For the pH condition for measuring the binding activity between anFcγR-binding domain contained in an antibody or Fc region (variant) andFcγR, an acidic or neutral pH condition may suitably be used. For thetemperature used in the measurement conditions, the binding activity(binding affinity) between an FcγR-binding domain and FcγR may beassessed, for example, at any temperature between 10° C. to 50° C. Apreferred temperature for determining the binding activity (bindingaffinity) of a human FcγR-binding domain to FcγR is, for example, 15° C.to 40° C. More preferably, to determine the binding activity (bindingaffinity) between an FcγR-binding domain and FcγR, any temperature from20° C. to 35° C., for example, such as any one of 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35° C. may be used. Anon-limiting example of such temperature is 25° C.

In one embodiment, where an antibody of Disclosure A or B has a constantregion (that may be modified), the constant region may have an Fc regionor an Fc region variant (preferably, a human Fc region or a human Fcregion variant), and preferably has an FcγR-binding domain within thescope of Disclosure A and an FcRn-binding domain within the scope ofDisclosures A and B described herein.

In one embodiment, where an antibody of Disclosure A has FcγR-bindingactivity, it may have an FcγR-binding domain, preferably a humanFcγR-binding domain. The FcγR-binding domain is not particularly limitedas long as the antibody has binding activity to or affinity for FcγR atacidic pH and/or neutral pH, and it may be a domain that has an activityto directly or indirectly bind FcγR.

In one embodiment, where an antibody of Disclosure A has FcγR-bindingactivity, it is preferable that the FcγR-binding activity of theantibody under a neutral pH condition is increased as compared to thatof a reference antibody which contains a native IgG constant region.From the perspective of comparing the FcγR-binding activity between thetwo, it is preferable, without limitations, that the antibody ofDisclosure A and the reference antibody which contains a native IgGconstant region have identical amino acid sequences in regions (forexample, the variable region) other than, preferably, the constantregion of the antibody of Disclosure A which has been modified at one ormore amino acid residues.

In one embodiment, where an antibody of Disclosure A has an FcγR-bindingactivity or an increased FcγR-binding activity under a neutral pHcondition (e.g., pH 7.4), without being bound by a theory, the antibodyis thought to possess the following properties in combination: theproperty of being shuttled between plasma and cellular endosome andrepeatedly binding to multiple antigens as a single antibody molecule byhaving an ion concentration-dependent antigen-binding domain; theproperty of being rapidly taken up into cells by having an increased pIand increased positive charge in the overall antibody; and the propertyof being rapidly taken up into cells by having an increased FcγR-bindingactivity under a neutral pH condition. As a result, the antibodyhalf-life in plasma can be further shortened, or the binding activity ofthe antibody toward the extracellular matrix can be further increased,or antigen elimination from plasma can be further promoted; thus theantibody of Disclosure A is beneficial. Those of ordinary skill in theart can routinely determine an optimal pI value for the antibody to takeadvantage of these properties.

In one embodiment, an FcγR-binding domain whose FcγR-binding activity ishigher than that of the Fc region or constant region of a native humanIgG in which the sugar chain linked at position 297 according to EUnumbering is a fucose-containing sugar chain can be produced bymodifying amino acid residues in the Fc region or constant region of anative human IgG (see WO2013/047752). Furthermore, a domain of anystructure that binds to FcγR can be used as an FcγR-binding domain. Inthis case, the FcγR-binding domain can be produced without the need tointroduce an amino acid modification, and alternatively, its affinityfor FcγR may be increased by introducing an additional modification.Such FcγR-binding domains can include Fab fragment antibodies that bindto FcγRIIIa, camel-derived single domain antibodies, and single chain Fvantibodies described in Schlapschly et al. (Protein Eng. Des. Sel. 22(3):175-188 (2009), Behar et al. (Protein Eng. Des. Sel. 21(1):1-10(2008)), and Kipriyanov et al., J Immunol. 169(1):137-144 (2002), andthe FcγRI-binding cyclic peptide described in Bonetto et al., FASEB J23(2):575-585 (2008). Whether the FcγR-binding activity of anFcγR-binding domain is higher than that of the Fc region or constantregion of a native human IgG in which the sugar chain linked at position297 according to EU numbering is a fucose-containing sugar chain can beappropriately assessed using the methods described above.

In one embodiment of Disclosure A, the starting FcγR-binding domainpreferably includes, for example, (human) IgG Fc region or (human) IgGconstant region. As long as a variant of the starting Fc region or thestarting constant region can bind to human FcγR in a neutral pH range,any Fc region or constant region can be used as the starting Fc regionor starting constant region. An Fc region or constant region obtained byfurther modifying a starting Fc region or starting constant region whoseamino acid residue(s) has been already modified from an Fc region orconstant region can also be appropriately used as the Fc region orconstant region of Disclosure A. A starting Fc region or startingconstant region may refer to the polypeptide itself, a compositioncontaining the starting Fc region or starting constant region, or anamino acid sequence encoding the starting Fc region or starting constantregion. The starting Fc region or starting constant region may includeknown Fc regions or known constant regions produced by recombinationtechnologies. The origin of the starting Fc region or starting constantregion is not limited, and it can be obtained from any organism ofnonhuman animals or from a human. Furthermore, the starting FcγR-bindingdomain can be obtained from cynomolgus monkeys, marmosets, Rhesusmonkeys, chimpanzees, or humans. The starting Fc region or startingconstant region can be preferably obtained from human IgG1; however, itis not limited to a particular IgG class. This means that the Fc regionof human IgG1, IgG2, IgG3, or IgG4 can be used as an appropriatestarting FcγR-binding domain, and it also means that within the scope ofDisclosure A described herein, an Fc region or constant region of an IgGclass or subclass derived from any organism can be preferably used asthe starting Fc region or starting constant region. Examples of a nativeIgG variant or modified form are described in publicly known literaturesuch as Strohl, Curr. Opin. Biotechnol. 20(6):685-691 (2009); Presta,Curr. Opin. Immunol. 20(4):460-470 (2008); Davis et al., Protein Eng.Des. Sel. 23(4):195-202 (2010); WO2009/086320, WO2008/092117;WO2007/041635; and WO2006/105338, but not limited thereto.

In one embodiment, amino acid residues of the starting FcγR-bindingdomain, starting Fc region, or starting constant region may contain, forexample, one or more mutations: for example, substitutions with aminoacid residues that are different from those in the starting Fc region orstarting constant region; insertions of one or more amino acid residuesinto the amino acid residues in the starting Fc region or startingconstant region; or deletions of one or more amino acid residues fromthose of the starting Fc region or starting constant region. The aminoacid sequences of Fc regions or constant regions after modifications arepreferably amino acid sequences containing at least a portion of an Fcregion or constant region that may not occur naturally. Such variantsnecessarily have a sequence identity or similarity of less than 100% tothe starting Fc regions or starting constant regions. For example, thevariants have an amino acid sequence identity or similarity of about 75%to less than 100%, more preferably about 80% to less than 100%, evenmore preferably about 85% to less than 100%, still more preferably about90% to less than 100%, and yet more preferably about 95% to less than100% to the amino acid sequence of the starting Fc region or startingconstant region. In a non-limiting example, at least one amino acid isdifferent between a modified Fc region or constant region of DisclosureA and the starting Fc region or starting constant region.

In one embodiment, an Fc region or constant region that has FcγR-bindingactivity in an acidic pH range and/or in a neutral pH range, which maybe contained in an antibody of Disclosure A, may be obtained by anymethod. Specifically, a variant of Fc region or constant region that hasFcγR-binding activity in a neutral pH range may be obtained by modifyingamino acids of a human IgG antibody which can be used as the starting Fcregion or starting constant region. IgG antibody Fc regions or IgGantibody constant regions suitable for modification can include, forexample, the Fc regions or constant regions of human IgG (IgG1, IgG2,IgG3, or IgG4, or variants thereof), and mutants spontaneously generatedtherefrom. For the Fc regions or constant regions of human IgG1, humanIgG2, human IgG3, or human IgG4 antibodies, a number of allotypesequences due to genetic polymorphism are described in “Sequences ofproteins of immunological interest”, NIH Publication No. 91-3242, andany of them may be used in Disclosure A. In particular, for the humanIgG1 sequence, the amino acid sequence of positions 356 to 358 accordingto EU numbering may be DEL or EEM.

In a further embodiment within the scope of Disclosure A, themodification into other amino acids is not limited as long as thevariants have an FcγR-binding activity in a neutral pH range. Amino acidposition(s) of such modification are reported, for example, in:WO2007/024249, WO2007/021841, WO2006/031370, WO2000/042072,WO2004/029207, WO2004/099249, WO2006/105338, WO2007/041635,WO2008/092117, WO2005/070963, WO2006/020114, WO2006/116260,WO2006/023403, WO2013/047752, WO2006/019447, WO2012/115241,WO2013/125667, WO2014/030728, WO2014/163101, WO2013/118858, andWO2014/030750.

Sites of amino acid modification in the constant region or Fc region toincrease the FcγR-binding activity in a neutral pH range can include,for example, one or more positions selected from the group consisting ofposition: 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250,251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284,285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339,376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434,436, and 440, according to EU numbering in the Fc region or constantregion of a human IgG antibody, as described in WO2013/047752.Modification of such amino acid residue may increase the FcγR binding ofthe Fc region or constant region of an IgG antibody under a neutral pHcondition. WO2013/047752 describes, as preferred modifications in anIgG-type constant region or Fc region, for example, modification of oneor more amino acid residues selected from the group consisting of: theamino acid at position 221 to either Lys or Tyr; the amino acid atposition 222 to any one of Phe, Trp, Glu, and Tyr; the amino acid atposition 223 to any one of Phe, Trp, Glu, and Lys; the amino acid atposition 224 to any one of Phe, Trp, Glu, and Tyr; the amino acid atposition 225 to any one of Glu, Lys, and Trp; the amino acid at position227 to any one of Glu, Gly, Lys, and Tyr; the amino acid at position 228to any one of Glu, Gly, Lys, and Tyr; the amino acid at position 230 toany one of Ala, Glu, Gly, and Tyr; the amino acid at position 231 to anyone of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 232 toany one of Glu, Gly, Lys, and Tyr; the amino acid at position 233 to anyone of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr, Val, Trp, and Tyr; the amino acid at position 234 to any one ofAla, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, and Tyr; the amino acid at position 235 to any one ofAla, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, and Tyr; the amino acid at position 236 to any one ofAla, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, and Tyr; the amino acid at position 237 to any one ofAsp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp, and Tyr; the amino acid at position 238 to any one of Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp, and Tyr; the amino acid at position 239 to any one of Asp, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp,and Tyr; the amino acid at position 240 to any one of Ala, Ile, Met, andThr; the amino acid at position 241 to any one of Asp, Glu, Leu, Arg,Trp, and Tyr; the amino acid at position 243 to any one of Glu, Leu,Gln, Arg, Trp, and Tyr; the amino acid at position 244 to His; the aminoacid at position 245 to Ala; the amino acid at position 246 to any oneof Asp, Glu, His, and Tyr; the amino acid at position 247 to any one ofAla, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr; the amino acid atposition 249 to any one of Glu, His, Gln, and Tyr; the amino acid atposition 250 to either Glu or Gln; the amino acid at position 251 toPhe; the amino acid at position 254 to any one of Phe, Met, and Tyr; theamino acid at position 255 to any one of Glu, Leu, and Tyr; the aminoacid at position 256 to any one of Ala, Met, and Pro; the amino acid atposition 258 to any one of Asp, Glu, His, Ser, and Tyr; the amino acidat position 260 to any one of Asp, Glu, His, and Tyr; the amino acid atposition 262 to any one of Ala, Glu, Phe, Ile, and Thr; the amino acidat position 263 to any one of Ala, Ile, Met, and Thr; the amino acid atposition 264 to any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position265 to any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 266 toany one of Ala, Ile, Met, and Thr; the amino acid at position 267 to anyone of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val, Trp, and Tyr; the amino acid at position 268 to any one of Asp,Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp; theamino acid at position 269 to any one of Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid atposition 270 to any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln,Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 271 to any oneof Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr, Val, Trp, and Tyr; the amino acid at position 272 to any one ofAsp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp,and Tyr; the amino acid at position 273 to either Phe or Ile; the aminoacid at position 274 to any one of Asp, Glu, Phe, Gly, His, Ile, Leu,Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid atposition 275 to either Leu or Trp; the amino acid at position 276 to anyone of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val,Trp, and Tyr; the amino acid at position 278 to any one of Asp, Glu,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, andTrp; the amino acid at position 279 to Ala; the amino acid at position280 to any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr; theamino acid at position 281 to any one of Asp, Lys, Pro, and Tyr; theamino acid at position 282 to any one of Glu, Gly, Lys, Pro, and Tyr;the amino acid at position 283 to any one of Ala, Gly, His, Ile, Lys,Leu, Met, Pro, Arg, and Tyr; the amino acid at position 284 to any oneof Asp, Glu, Leu, Asn, Thr, and Tyr; the amino acid at position 285 toany one of Asp, Glu, Lys, Gln, Trp, and Tyr; the amino acid at position286 to any one of Glu, Gly, Pro, and Tyr; the amino acid at position 288to any one of Asn, Asp, Glu, and Tyr; the amino acid at position 290 toany one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr; the aminoacid at position 291 to any one of Asp, Glu, Gly, His, Ile, Gln, andThr; the amino acid at position 292 to any one of Ala, Asp, Glu, Pro,Thr, and Tyr; the amino acid at position 293 to any one of Phe, Gly,His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; theamino acid at position 294 to any one of Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid atposition 295 to any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn,Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 296 toany one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, and Val; the amino acid at position 297 to any one of Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val,Trp, and Tyr; the amino acid at position 298 to any one of Ala, Asp,Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; theamino acid at position 299 to any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; theamino acid at position 300 to any one of Ala, Asp, Glu, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; the aminoacid at position 301 to any one of Asp, Glu, His, and Tyr; the aminoacid at position 302 to Ile; the amino acid at position 303 to any oneof Asp, Gly, and Tyr; the amino acid at position 304 to any one of Asp,His, Leu, Asn, and Thr; the amino acid at position 305 to any one ofGlu, Ile, Thr, and Tyr; the amino acid at position 311 to any one ofAla, Asp, Asn, Thr, Val, and Tyr; the amino acid at position 313 to Phe;the amino acid at position 315 to Leu; the amino acid at position 317 toeither Glu or Gln; the amino acid at position 318 to any one of His,Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; the amino acid at position320 to any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val,Trp, and Tyr; the amino acid at position 322 to any one of Ala, Asp,Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid atposition 323 to Ile; the amino acid at position 324 to any one of Asp,Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr; theamino acid at position 325 to any one of Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; theamino acid at position 326 to any one of Ala, Asp, Glu, Gly, Ile, Leu,Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; the amino acid atposition 327 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid at position328 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 329 toany one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr, Val, Trp, and Tyr; the amino acid at position 330 to any oneof Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr,Val, Trp, and Tyr; the amino acid at position 331 to any one of Asp,Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr; the aminoacid at position 332 to any one of Ala, Asp, Glu, Phe, Gly, His, Lys,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; the aminoacid at position 333 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile,Leu, Met, Pro, Ser, Thr, Val, and Tyr; the amino acid at position 334 toany one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr; the amino acid atposition 335 to any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,Arg, Ser, Val, Trp, and Tyr; the amino acid at position 336 to any oneof Glu, Lys, and Tyr; the amino acid at position 337 to any one of Glu,His, and Asn; the amino acid at position 339 to any one of Asp, Phe,Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr; the amino acid atposition 376 to either Ala or Val; the amino acid at position 377 toeither Gly or Lys; the amino acid at position 378 to Asp; the amino acidat position 379 to Asn; the amino acid at position 380 to any one ofAla, Asn, and Ser; the amino acid at position 382 to either Ala or Ile;the amino acid at position 385 to Glu; the amino acid at position 392 toThr; the amino acid at position 396 to Leu; the amino acid at position421 to Lys; the amino acid at position 427 to Asn; the amino acid atposition 428 to either Phe or Leu; the amino acid at position 429 toMet; the amino acid at position 434 to Trp; the amino acid at position436 to Ile; and the amino acid at position 440 to any one of Gly, His,Ile, Leu, and Tyr, according to EU numbering. The number of amino acidsto be modified is not particularly limited, and it is possible to modifyan amino acid at only one position or amino acids at two or morepositions. Combinations of amino acid modifications at two or morepositions are shown in Table 5 of WO2013/047752. Modification of theseamino acid residues can also be appropriately introduced into theantibodies of Disclosure A.

In one embodiment, the binding activity of (the FcγR-binding domain of)the antibody of Disclosure A toward (human) FcγR(s), such as any one ormore of FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, may be higherthan that of (the Fc region or constant region of) a native IgG or thatof a reference antibody containing the starting Fc region or startingconstant region. For example, the FcγR-binding activity of (theFcγR-binding domain of) an antibody of Disclosure A may be 55% or more,60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 100% or more, 105% or more, preferably110% or more, 115% or more, 120% or more, 125% or more, particularlypreferably 130% or more, 135% or more, 140% or more, 145% or more, 150%or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% ormore, 180% or more, 185% or more, 190% or more, or 195% or more ascompared to the FcγR-binding activity of the reference antibody, or2-fold or more, 2.5-fold or more, 3-fold or more, 3.5-fold or more,4-fold or more, 4.5-fold or more, 5-fold or more, 7.5-fold or more,10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more,50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more,90-fold or more, or 100-fold or more greater than the FcγR-bindingactivity of the reference antibody.

In a further embodiment, the level of increase in the binding activityto an inhibitory FcγR (FcγRIIb-1 and/or FcγRIIb-2) (in a neutral pHrange) may be greater than the level of increase in the binding activityto an activating FcγR (FcγRIa: FcγRIb; FcγRIc; FcγRIIIa includingallotype V158; FcγRIIIa including allotype F158; FcγRIIIb includingallotype FcγRIIIb-NA1; FcγRIIIb including allotype FcγRIIIb-NA2; FcγRIIaincluding allotype H131; or FcγRIIa including allotype R131).

In one embodiment, an antibody of Disclosure A may have binding activityto FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

In one embodiment, preferred FcγR-binding domains of Disclosure A alsoinclude, for example, FcγR-binding domains whose binding activity to aspecific FcγR is greater than the binding activity to other FcγR(FcγR-binding domains having a selective FcγR-binding activity). Wherean antibody (or the Fc region as the FcγR-binding domain) is used, asingle antibody molecule can bind only to a single FcγR molecule. Thus,a single antibody molecule in a state bound to an inhibitory FcγR cannotbind to other activating FcγRs, and a single antibody molecule in astate bound to an activating FcγR cannot bind to other activating FcγRsor inhibitory FcγRs.

As described above, an activating FcγR preferably includes, for example,FcγRI (CD64) such as FcγRIa, FcγRIb, or FcγRIc; and FcγRIII (CD16) suchas FcγRIIIa (such as allotype V158 or F158) or FcγRIIIb (such asallotype FcγRIIIb-NA1 or FcγRIIIb-NA2). Meanwhile, an inhibitory FcγRpreferably includes, for example, FcγRIIb (such as FcγRIIb-1 orFcγRIIb-2).

In one embodiment, FcγR-binding domains that have a greater bindingactivity to inhibitory FcγR than to activating FcγR can be used as theselective FcγR-binding domain contained in an antibody of Disclosure A.Such selective FcγR-binding domains can include, for example,FcγR-binding domains that have a greater binding activity to FcγRIIb(such as FcγRIIb-1 and/or FcγRIIb-2) than to any one or more ofactivating FcγR selected from the group consisting of: FcγRI (CD64) suchas FcγRIa, FcγRIb, or FcγRIc; FcγRIII (CD16) such as FcγRIIIa (such asallotype V158 or F158) or FcγRIIIb (such as FcγRIIIb-NA1 orFcγRIIIb-NA2); FcγRII (CD32) such as FcγRIIa (including allotype H131 orR131); and FcγRIIc.

Furthermore, whether an FcγR-binding domain has a selective bindingactivity can be assessed by comparing the binding activity to each FcγRdetermined by the methods described above, for example, by comparing thevalue (ratio) obtained by dividing the KD value for activating FcγR bythe KD value for inhibitory FcγR, more specifically by comparing theFcγR selectivity index shown in Equation 1 below:FcγR selectivity index=KD value for activating FcγR/KD value forinhibitory FcγR  [Equation 1]:

In Equation 1, the KD value for activating FcγR refers to the KD valuefor one or more of: FcγRIa; FcγRIb; FcγRIc; FcγRIIIa including allotypeV158 and/or F158; FcγRIIIb including FcγRIIIb-NA1 and/or FcγRIIIb-NA2;FcγRIIa including allotype H131 and/or R131; and FcγRIIc; and the KDvalue for inhibitory FcγR refers to the KD value for FcγRIIb-1 and/orFcγRIIb-2. The activating FcγR and inhibitory FcγR for use indetermining the KD values may be selected in any combination. Forexample, it is possible to use a value (ratio) determined by dividingthe KD value for FcγRIIa including allotype H131 by the KD value forFcγRIIb-1 and/or FcγRIIb-2, without limitations thereto.

The FcγR selectivity index can be, for example: 1.2 or greater, 1.3 orgreater, 1.4 or greater, 1.5 or greater, 1.6 or greater, 1.7 or greater,1.8 or greater, 1.9 or greater, 2 or greater, 3 or greater, 5 orgreater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 orgreater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 35or greater, 40 or greater, 45 or greater, 50 or greater, 55 or greater,60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 orgreater, 85 or greater, 90 or greater, 95 or greater, 100 or greater,110 or greater, 120 or greater, 130 or greater, 140 or greater, 150 orgreater, 160 or greater, 170 or greater, 180 or greater, 190 or greater,200 or greater, 210 or greater, 220 or greater, 230 or greater, 240 orgreater, 250 or greater, 260 or greater, 270 or greater, 280 or greater,290 or greater, 300 or greater, 310 or greater, 320 or greater, 330 orgreater, 340 or greater, 350 or greater, 360 or greater, 370 or greater,380 or greater, 390 or greater, 400 or greater, 410 or greater, 420 orgreater, 430 or greater, 440 or greater, 450 or greater, 460 or greater,470 or greater, 480 or greater, 490 or greater, 500 or greater, 520 orgreater, 540 or greater, 560 or greater, 580 or greater, 600 or greater,620 or greater, 640 or greater, 660 or greater, 680 or greater, 700 orgreater, 720 or greater, 740 or greater, 760 or greater, 780 or greater,800 or greater, 820 or greater, 840 or greater, 860 or greater, 880 orgreater, 900 or greater, 920 or greater, 940 or greater, 960 or greater,980 or greater, 1000 or greater, 1500 or greater, 2000 or greater, 2500or greater, 3000 or greater, 3500 or greater, 4000 or greater, 4500 orgreater, 5000 or greater, 5500 or greater, 6000 or greater, 6500 orgreater, 7000 or greater, 7500 or greater, 8000 or greater, 8500 orgreater, 9000 or greater, 9500 or greater, 10000 or greater, or 100000or greater; but it is not limited thereto.

In one embodiment, (an antibody containing) an Fc region variant orconstant region variant in which the amino acid at position 238 or 328,according to EU numbering of human IgG (IgG1, IgG2, IgG3, or IgG4) isAsp or Glu, respectively, can be preferably used as antibodies ofDisclosure A containing an Fc region variant or constant region variant,since as specifically described in WO2013/125667, WO2012/115241, andWO2013/047752, it has a greater binding activity to FcγRIIb-1 and/orFcγRIIb-2 than to FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including allotypeV158, FcγRIIIa including allotype F158, FcγRIIIb including allotypeFcγRIIIb-NA1, FcγRIIIb including allotype FcγRIIIb-NA2, FcγRIIaincluding allotype H131, FcγRIIa including allotype R131, and/orFcγRIIc. In such an embodiment, the antibodies of Disclosure A havebinding activity to all activating FcγRs (herein, which are selectedfrom the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa FcγRIIIbFcγRIIa) and FcγRIIb, and their FcγRIIb-binding activity is maintainedor increased, and/or their binding activity to all activating FcγRs isreduced, as compared to the reference antibody that contains a nativeIgG constant region or a native IgG Fc region.

In one embodiment for the antibodies of Disclosure A containing an Fcregion variant or constant region variant, their binding activity toFcγRIIb may be maintained or increased and their binding activity toFcγRIIa (type H) and FcγRIIa (type R) may be reduced as compared tothose of a reference antibody having the constant region or Fc region ofa native IgG. Such antibodies may have increased binding selectivity toFcγRIIb over FcγRIIa.

Within the scope of Disclosure A described herein, the extent that the“binding activity to all activating FcγRs is reduced” can be, but is notlimited to, 99% or less, 98% or less, 97% or less, 96% or less, 95% orless, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less,88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% orless, 76% or less, 74% or less, 72% or less, 70% or less, 68% or less,66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% orless, 54% or less, 52% or less, 50% or less, 45% or less, 40% or less,35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% orless, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5%or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05%or less, 0.01% or less, or 0.005% or less.

Within the scope of Disclosure A described herein, the extent that the“FcγRIIb-binding activity is maintained or increased”, the “bindingactivity to FcγRIIb is maintained or increased”, or the “maintained orincreased binding activity to FcγRIIb” can be, but is not limited to,55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% orgreater, 80% or greater, 85% or greater, 87% or greater, 88% or greater,89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% orgreater, 94% or greater, 95% or greater, 96% or greater, 97% or greater,98% or greater, 99% or greater, 99.5% or greater, 100% or greater, 101%or greater, 102% or greater, 103% or greater, 104% or greater, 105% orgreater, 106% or greater, 107% or greater, 108% or greater, 109% orgreater, 110% or greater, 112% or greater, 114% or greater, 116% orgreater, 118% or greater, 120% or greater, 122% or greater, 124% orgreater, 126% or greater, 128% or greater, 130% or greater, 132% orgreater, 134% or greater, 136% or greater, 138% or greater, 140% orgreater, 142% or greater, 144% or greater, 146% or greater, 148% orgreater, 150% or greater, 155% or greater, 160% or greater, 165% orgreater, 170% or greater, 175% or greater, 180% or greater, 185% orgreater, 190% or greater, 195% or greater, 2-fold or greater, 3-fold orgreater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 7-foldor greater, 8-fold or greater, 9-fold or greater, 10-fold or greater,20-fold or greater, 30-fold or greater, 40-fold or greater, 50-fold orgreater, 60-fold or greater, 70-fold or greater, 80-fold or greater,90-fold or greater, 100-fold or greater, 200-fold or greater, 300-foldor greater, 400-fold or greater, 500-fold or greater, 600-fold orgreater, 700-fold or greater, 800-fold or greater, 900-fold or greater,1000-fold or greater, 10000-fold or greater, or 100000-fold or greater.

Within the scope of Disclosure A described herein, the extent that the“binding activity to FcγRIIa (type H) and FcγRIIa (type R) is reduced”or the “reduced binding activity to FcγRIIa (type H) and FcγRIIa (typeR)” can be, but is not limited to, 99% or less, 98% or less, 97% orless, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less,91% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% orless, 80% or less, 78% or less, 76% or less, 74% or less, 72% or less,70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% orless, 58% or less, 56% or less, 54% or less, 52% or less, 50% or less,45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% orless, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2%or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% orless, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

Within the scope of Disclosure A described herein, modifications thatincrease binding selectivity to FcγRIIb over FcγRIIa (type R) may bepreferred, and modifications that increase binding selectivity toFcγRIIb over FcγRIIa (type H) may be more preferred, and as reported inWO2013/047752, preferred amino acid substitutions for such modificationsmay include, for example, according to EU numbering: (a) modification bysubstituting Gly at position 237 with Trp; (b) modification bysubstituting Gly at position 237 with Phe; (c) modification bysubstituting Pro at position 238 with Phe; (d) modification bysubstituting Asn at position 325 with Met; (e) modification bysubstituting Ser at position 267 with Ile; (f) modification bysubstituting Leu at position 328 with Asp; (g) modification bysubstituting Ser at position 267 with Val; (h) modification bysubstituting Leu at position 328 with Trp; (i) modification bysubstituting Ser at position 267 with Gln; (j) modification bysubstituting Ser at position 267 with Met; (k) modification bysubstituting Gly at position 236 with Asp; (1) modification bysubstituting Ala at position 327 with Asn; (m) modification bysubstituting Asn at position 325 with Ser; (n) modification bysubstituting Leu at position 235 with Tyr; (o) modification bysubstituting Val at position 266 with Met; (p) modification bysubstituting Leu at position 328 with Tyr; (q) modification bysubstituting Leu at position 235 with Trp; (r) modification bysubstituting Leu at position 235 with Phe; (s) modification bysubstituting Ser at position 239 with Gly; (t) modification bysubstituting Ala at position 327 with Glu; (u) modification bysubstituting Ala at position 327 with Gly; (v) modification bysubstituting Pro at position 238 with Leu; (w) modification bysubstituting Ser at position 239 with Leu; (x) modification bysubstituting Leu at position 328 with Thr; (y) modification bysubstituting Leu at position 328 with Ser; (z) modification bysubstituting Leu at position 328 with Met; (aa) modification bysubstituting Pro at position 331 with Trp; (ab) modification bysubstituting Pro at position 331 with Tyr; (ac) modification bysubstituting Pro at position 331 with Phe; (ad) modification bysubstituting Ala at position 327 with Asp; (ae) modification bysubstituting Leu at position 328 with Phe; (af) modification bysubstituting Pro at position 271 with Leu; (ag) modification bysubstituting Ser at position 267 with Glu; (ah) modification bysubstituting Leu at position 328 with Ala; (ai) modification bysubstituting Leu at position 328 with Ile; (aj) modification bysubstituting Leu at position 328 with Gln; (ak) modification bysubstituting Leu at position 328 with Val; (al) modification bysubstituting Lys at position 326 with Trp; (am) modification bysubstituting Lys at position 334 with Arg; (an) modification bysubstituting His at position 268 with Gly; (ao) modification bysubstituting His at position 268 with Asn; (ap) modification bysubstituting Ser at position 324 with Val; (aq) modification bysubstituting Val at position 266 with Leu; (ar) modification bysubstituting Pro at position 271 with Gly; (as) modification bysubstituting Ile at position 332 with Phe; (at) modification bysubstituting Ser at position 324 with Ile; (au) modification bysubstituting Glu at position 333 with Pro; (av) modification bysubstituting Tyr at position 300 with Asp; (aw) modification bysubstituting Ser at position 337 with Asp; (ax) modification bysubstituting Tyr at position 300 with Gln; (ay) modification bysubstituting Thr at position 335 with Asp; (az) modification bysubstituting Ser at position 239 with Asn; (ba) modification bysubstituting Lys at position 326 with Leu; (bb) modification bysubstituting Lys at position 326 with Ile; (bc) modification bysubstituting Ser at position 239 with Glu; (bd) modification bysubstituting Lys at position 326 with Phe; (be) modification bysubstituting Lys at position 326 with Val; (bf) modification bysubstituting Lys at position 326 with Tyr; (bg) modification bysubstituting Ser at position 267 with Asp; (bh) modification bysubstituting Lys at position 326 with Pro; (bi) modification bysubstituting Lys at position 326 with His; (bj) modification bysubstituting Lys at position 334 with Ala; (bk) modification bysubstituting Lys at position 334 with Trp; (bl) modification bysubstituting His at position 268 with Gln; (bm) modification bysubstituting Lys at position 326 with Gln; (bn) modification bysubstituting Lys at position 326 with Glu; (bo) modification bysubstituting Lys at position 326 with Met; (bp) modification bysubstituting Val at position 266 with Ile; (bq) modification bysubstituting Lys at position 334 with Glu; (br) modification bysubstituting Tyr at position 300 with Glu; (bs) modification bysubstituting Lys at position 334 with Met; (bt) modification bysubstituting Lys at position 334 with Val; (bu) modification bysubstituting Lys at position 334 with Thr; (bv) modification bysubstituting Lys at position 334 with Ser; (bw) modification bysubstituting Lys at position 334 with His; (bx) modification bysubstituting Lys at position 334 with Phe; (by) modification bysubstituting Lys at position 334 with Gln; (bz) modification bysubstituting Lys at position 334 with Pro; (ca) modification bysubstituting Lys at position 334 with Tyr; (cb) modification bysubstituting Lys at position 334 with Ile; (cc) modification bysubstituting Gln at position 295 with Leu; (cd) modification bysubstituting Lys at position 334 with Leu; (ce) modification bysubstituting Lys at position 334 with Asn; (cf) modification bysubstituting His at position 268 with Ala; (cg) modification bysubstituting Ser at position 239 with Asp; (ch) modification bysubstituting Ser at position 267 with Ala; (ci) modification bysubstituting Leu at position 234 with Trp; (cj) modification bysubstituting Leu at position 234 with Tyr; (ck) modification bysubstituting Gly at position 237 with Ala; (cl) modification bysubstituting Gly at position 237 with Asp; (cm) modification bysubstituting Gly at position 237 with Glu; (cn) modification bysubstituting Gly at position 237 with Leu; (co) modification bysubstituting Gly at position 237 with Met; (cp) modification bysubstituting Gly at position 237 with Tyr; (cq) modification bysubstituting Ala at position 330 with Lys; (cr) modification bysubstituting Ala at position 330 with Arg; (cs) modification bysubstituting Glu at position 233 with Asp; (ct) modification bysubstituting His at position 268 with Asp; (cu) modification bysubstituting His at position 268 with Glu; (cv) modification bysubstituting Lys at position 326 with Asp; (cw) modification bysubstituting Lys at position 326 with Ser; (cx) modification bysubstituting Lys at position 326 with Thr; (cy) modification bysubstituting Val at position 323 with Ile; (cz) modification bysubstituting Val at position 323 with Leu; (da) modification bysubstituting Val at position 323 with Met; (db) modification bysubstituting Tyr at position 296 with Asp; (dc) modification bysubstituting Lys at position 326 with Ala; (dd) modification bysubstituting Lys at position 326 with Asn; and (de) modification bysubstituting Ala at position 330 with Met.

The modifications described above may be at a single position alone orat two or more positions in combination. Alternatively, such preferredmodifications may include, for example, those shown in Tables 14 to 15,17 to 24, and 26 to 28 of WO2013/047752, for example, variants of humanconstant region or human Fc region, in which the amino acid at position238 according to EU numbering is Asp and the amino acid at position 271according to EU numbering is Gly in human IgG (IgG1, IgG2, IgG3, orIgG4); in addition, one or more of position(s) 233, 234, 237, 264, 265,266, 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333, and 396,according to EU numbering may be substituted. In this case, the variantsmay include, but are not limited to, variants of human constant regionor human Fc region that contain one or more of:

Asp at position 233, Tyr at position 234, Asp at position 237, Ile atposition 264, Glu at position 265, any one of Phe, Met, and Leu atposition 266, any one of Ala, Glu, Gly, and Gln at position 267, eitherAsp or Glu at position 268, Asp at position 269, any one of Asp, Phe,Ile, Met, Asn, and Gln at position 272, Asp at position 296, either Alaor Asp at position 326, Gly at position 327, either Lys or Arg atposition 330, Ser at position 331, Thr at position 332, any one of Thr,Lys, and Arg at position 333, and any one of Asp, Glu, Phe, Ile, Lys,Leu, Met, Gln, Arg, and Tyr at position 396, according to EU numbering.

In an alternative embodiment, antibodies of Disclosure A containing anFc region variant or constant region variant may have maintained orincreased binding activity to FcγRIIb and reduced binding activity toFcγRIIa (type H) and FcγRIIa (type R) as compared to a referenceantibody containing the constant region or Fc region of a native IgG.Preferred sites of amino acid substitution for such variants may be, asreported in WO2014/030728, for example, the amino acid at position 238according to EU numbering and at least one amino acid position selectedfrom the group consisting of position 233, 234, 235, 237, 264, 265, 266,267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334,355, 356, 358, 396, 409, and 419, according to EU numbering.

More preferably, the variants may have Asp at position 238 according toEU numbering, and at least one amino acid selected from the amino acidgroup of: Asp at position 233, Tyr at position 234, Phe at position 235,Asp at position 237, Ile at position 264, Glu at position 265, Phe, Leu,or Met at position 266, Ala, Glu, Gly, or Gln at position 267, Asp, Gln,or Glu at position 268, Asp at position 269, Gly at position 271, Asp,Phe, Ile, Met, Asn, Pro, or Gln at position 272, Gln at position 274,Asp or Phe at position 296, Ala or Asp at position 326, Gly at position327, Lys, Arg, or Ser at position 330, Ser at position 331, Lys, Arg,Ser, or Thr at position 332, Lys, Arg, Ser, or Thr at position 333, Arg,Ser, or Thr at position 334, Ala or Gln at position 355, Glu at position356, Met at position 358, Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at position 396, Arg atposition 409, and Glu at position 419, according to EU numbering.

In an alternative embodiment, antibodies of Disclosure A containing anFc region variant or constant region variant may have maintained bindingactivity to FcγRIIb and reduced binding activity to all activatingFcγRs, FcγRIIa (type R) in particular, as compared to a referenceantibody containing the constant region or Fc region of a native IgG.Preferred sites of amino acid substitution for such variants may be, asreported in WO2014/163101, for example, in addition to the amino acid atposition 238 according to EU numbering), at least one amino acidposition selected from positions 235, 237, 241, 268, 295, 296, 298, 323,324, and 330, according to EU numbering. More preferably, the variantsmay have Asp at position 238 according to EU numbering, and at least oneamino acid selected from the amino acid group of: Phe at position 235;Gln or Asp at position 237; Met or Leu at position 241; Pro at position268; Met or Val at position 295; Glu, His, Asn, or Asp at position 296;Ala or Met at position 298; Ile at position 323; Asn or His at position324; and His or Tyr at position 330, according to EU numbering.

Within the scope of Disclosure A described herein, the level of the“maintained binding activity to FcγRIIb” can be, but is not limited to,55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% orgreater, 80% or greater, 81% or greater, 82% or greater, 83% or greater,84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% orgreater, 89% or greater, 90% or greater, 91% or greater, 92% or greater,93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% orgreater, 98% or greater, 99% or greater, 99.5% or greater, 100% orgreater, 101% or greater, 102% or greater, 103% or greater, 104% orgreater, 105% or greater, 106% or greater, 107% or greater, 108% orgreater, 109% or greater, 110% or greater, 120% or greater, 130% orgreater, 140% or greater, 150% or greater, 175% or greater, or 2-fold orgreater.

Within the scope of Disclosure A described herein, the level of theaforementioned “reduced binding activity to all activating FcγRs,FcγRIIa (type R) in particular” can be, but is not limited to, 74% orless, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less,62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% orless, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less,25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% orless, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less,0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less,or 0.005% or less.

WO2014/030750 also reports variants of the mouse constant region and Fcregion. In an embodiment, antibodies of Disclosure A or B may comprisesuch a variant.

Within the scope of Disclosures A and B described herein, unlike FcγRwhich belongs to the immunoglobulin superfamily, “FcRn”, in particularhuman FcRn, is structurally similar to polypeptides of majorhistocompatibility complex (MEW) class I, and exhibits 22% to 29%sequence identity with MEW class I molecules (Ghetie et al., Immunol.Today 18(12), 592-598 (1997)). FcRn is expressed as a heterodimerconsisting of a soluble β or light chain (β2 microglobulin) complexedwith a transmembrane a or heavy chain. Like MHC, the α chain of FcRncontains three extracellular domains (α1, α2, and α3), and its shortcytoplasmic domain tethers proteins to the cell surface. α1 and α2domains interact with the FcRn-binding domain of the antibody Fc region(Raghavan et al., Immunity 1:303-315 (1994)).

FcRn is expressed in the maternal placenta and yolk sac of mammals, andis involved in mother-to-fetus IgG transfer. In addition, in the smallintestines of neonatal rodents where FcRn is expressed, FcRn is involvedin transfer of maternal IgG across brush border epithelium from ingestedcolostrum or milk. FcRn is expressed in a variety of other tissues andendothelial cell systems of various species. FcRn is also expressed inadult human vascular endothelia, muscle vascular system, and liversinusoidal capillaries. FcRn is believed to play a role in maintainingthe plasma IgG concentration by binding to IgG and recycling the IgG toserum. Typically, binding of FcRn to IgG molecules is strictly pHdependent. The optimal binding is observed in an acidic pH range below7.0.

The polynucleotide and amino acid sequences of human FcRn may bederived, for example, from the precursors shown in NM_004107.4 andNP_004098.1 (containing the signal sequence), respectively (RefSeqaccession numbers are shown in parentheses).

The precursors form complexes with human β2-microglobulin in vivo. Thus,by using known recombinant expression techniques, soluble human FcRncapable of forming a complex with human β2-microglobulin may be producedfor appropriate use in various experimental systems. Such soluble humanFcRn may be used to assess antibodies or Fc region variants for theirFcRn-binding activity. In Disclosure A or B, FcRn is not particularlylimited as long as it is in a form which can bind to the FcRn-bindingdomain; however, preferred FcRn may be human FcRn.

Within the scope of Disclosures A and B described herein, where anantibody or Fc region variant has FcRn-binding activity, it may have an“FcRn-binding domain”, preferably a human FcRn-binding domain. TheFcRn-binding domain is not particularly limited as long as the antibodyhas binding activity to or affinity for FcRn at an acidic pH and/or at aneutral pH; or it may be a domain that has the activity to directly orindirectly bind to FcRn. Such domains include, but are not limited to,the Fc regions of IgG-type immunoglobulins, albumin, albumin domain 3,anti-FcRn antibodies, anti-FcRn peptides, and anti-FcRn Scaffoldmolecules, which have the activity of directly binding to FcRn, andmolecules that bind to IgG or albumin, which have the activity ofbinding to FcRn indirectly. In Disclosure A or B, it is also possible touse domains that have FcRn-binding activity in an acidic pH range and/orin a neutral pH range. If the domains have FcRn-binding activity in anacidic pH range and/or in a neutral pH range originally, they can beused without further modification. If the domains have only a weak or noFcRn-binding activity in an acidic pH range and/or in a neutral pHrange, amino acid residues in the FcRn-binding domain of the antibody orFc region variant may be modified to have FcRn-binding activity in anacidic pH range and/or in a neutral pH range. Alternatively, amino acidsof domains that originally have FcRn-binding activity in an acidic pHrange and/or in a neutral pH range may be modified to further increasetheir FcRn-binding activity. The FcRn-binding activity in an acidic pHrange and/or in a neutral pH range can be compared before and afteramino acid modification to find amino acid modifications of interest forthe FcRn-binding domains.

FcRn-binding domains may be preferably regions that directly bind toFcRn. Such preferred FcRn-binding domains include, for example, constantregions and Fc regions of antibodies. However, regions capable ofbinding to a polypeptide having FcRn-binding activity, such as albuminand IgG, can indirectly bind to FcRn via albumin, IgG. Thus, theFcRn-binding regions may be regions that bind to a polypeptide that hasbinding activity to albumin or IgG. Without limitations, to promoteantigen elimination from plasma, FcRn-binding domains whose FcRn-bindingactivity is greater at a neutral pH are preferred, while to improveantibody retention in plasma, FcRn-binding domains whose FcRn-bindingactivity is greater at an acidic pH are preferred. For example, it ispossible to select FcRn-binding domains whose FcRn-binding activity isoriginally greater at a neutral pH or acidic pH. Alternatively, aminoacids of an antibody or Fc region variant may be modified to conferFcRn-binding activity at a neutral pH or acidic pH. Alternatively, it ispossible to increase the pre-existing FcRn-binding activity at a neutralpH or acidic pH.

Within the scope of Disclosures A and B described herein, whether theFcRn-binding activity of an antibody or Fc region (variant) isincreased, (substantially) maintained, or reduced as compared to that ofthe antibody or Fc region (variant) before modification can be assessedby known methods such as those described in the Examples herein, and forexample, BIACORE, Scatchard plot and flow cytometer (see WO2013/046722).The extracellular domain of human FcRn may be used as a soluble antigenin these assays. Those of ordinary skill in the art can appropriatelyselect the conditions besides pH in measuring the FcRn-binding activityof an antibody or Fc region (variant). The assay can be carried out, forexample, under the conditions of MES buffer and 37° C., as described inWO2009/125825. The FcRn-binding activity of an antibody or Fc region(variant) can be assessed, for example, by loading FcRn as an analyte onan antibody-immobilized chip.

The FcRn-binding activity of an antibody or Fc region (variant) can beassessed based on the dissociation constant (KD), apparent dissociationconstant (apparent KD), dissociation rate (kd), apparent dissociation(apparent kd).

As for the pH conditions for measuring the binding activity between FcRnand the FcRn-binding domain contained in an antibody or Fc region(variant), acidic pH condition or neutral pH condition may be suitablyused. As for the temperature conditions for measuring the bindingactivity (binding affinity) between FcRn and the FcRn-binding domain,any temperature of 10° C. to 50° C. may be used. To determine thebinding activity (binding affinity) between FcRn and the humanFcRn-binding domain, preferably a temperature of 15° C. to 40° C. may beused. More preferably, any temperature from 20° C. to 35° C. such as anyone of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and35° C. may be used. A non-limiting example of such temperature can be25° C.

In one embodiment, where antibodies of Disclosure A or B haveFcRn-binding activity, they may have an FcRn-binding domain, preferablya human FcRn-binding domain. The FcRn-binding domain is not particularlylimited as long as the antibodies have binding activity to or affinityfor FcRn at an acidic pH and/or a neutral pH, and it may be a domainthat has an activity of directly or indirectly binding to FcRn. In onespecific embodiment, it may be preferable that the antibody ofDisclosure A or B has, for example, an increased FcRn-binding activityunder a neutral pH condition as compared to a reference antibodycontaining the constant region of a native IgG (see WO2013/046722). Fromthe perspective of comparing the FcRn-binding activity between the two,it may be preferable that, without limitations, the antibody ofDisclosure A or B and the reference antibody containing the constantregion of a native IgG have identical amino acid sequences in theregions (for example, the variable region) other than, preferably, theconstant region of the antibody of Disclosure A or B which has beenmodified at one or more amino acid residues.

In one embodiment, within the scope of Disclosure A described herein,where an antibody of Disclosure A has an increased FcRn-binding activityunder a neutral pH condition, without being bound by a particulartheory, the antibody of Disclosure A may possess any two or more of thefollowing properties in combination: the property of being shuttledbetween plasma and cellular endosome and repeatedly binding to multipleantigens as a single antibody molecule by having an ionconcentration-dependent antigen-binding domain; the property of beingrapidly taken up into cells by having increased pI and increasedpositive charge in the overall antibody; and the property of beingrapidly taken up into cells by having an increased FcRn-binding activityunder a neutral pH condition. As a result, the antibody half-life inplasma can be further shortened, or the binding activity of the antibodytoward the extracellular matrix can be further increased, or antigenelimination from plasma can be further promoted. Those of ordinary skillin the art can determine an optimal pI value for the antibody ofDisclosure A to take advantage of these properties.

Within the scope of Disclosures A and B described herein, according tothe Yeung et al. (J. Immunol. 182:7663-7671 (2009)), the activity of anative human IgG1 to bind to human FcRn is KD 1.7 μM in an acidic pHrange (pH 6.0), whereas in a neutral pH range the activity is almostundetectable. Thus, to increase the FcRn-binding activity in a neutralpH range, it may preferable to use, as an antibody of Disclosure A or B:an antibody or a constant region variant or Fc region variant whosehuman FcRn-binding activity in an acidic pH range is KD 20 μM orstronger and whose human FcRn-binding activity in a neutral pH range iscomparable to or stronger than that of a native human IgG; preferably anantibody or a constant region variant or Fc region variant whose humanFcRn-binding activity in an acidic pH range is KD 2.0 μM or stronger andwhose human FcRn-binding activity in a neutral pH range is KD 40 μM orstronger; and more preferably an antibody or a constant region variantor Fc region variant whose human FcRn-binding activity in an acidic pHrange is KD 0.5 μM or stronger and whose human FcRn-binding activity ina neutral pH range is KD 15 μM or stronger. The KD values are determinedby the method described in Yeung et al. (J. Immunol. 182:7663-7671(2009) (by immobilizing an antibody onto a chip and loading human FcRnas an analyte)).

Within the scope of Disclosures A and B described herein, a domain ofany structure that binds to FcRn can be used as an FcRn-binding domain.In this case, the FcRn-binding domain can be produced without the needto introduce an amino acid modification, or the affinity for FcRn may beincreased by introducing an additional modification.

Within the scope of Disclosures A and B described herein, the startingFcRn-binding domain can include for example, the Fc region or constantregion of (human) IgG. As long as a variant of the starting Fc region orstarting constant region can bind to FcRn in an acidic pH range and/orin a neutral pH range, any Fc region or constant region can be used asthe starting Fc region or starting constant region. Or, an Fc region orconstant region obtained by further modifying a starting Fc region orstarting constant region whose amino acid residues have been alreadymodified from an Fc region or constant region can also be appropriatelyused as the Fc region or constant region. The starting Fc region orstarting constant region may include known Fc regions produced byrecombination. A starting Fc region or starting constant region mayrefer to the polypeptide itself, a composition containing the startingFc region or starting constant region, or an amino acid sequenceencoding the starting Fc region or starting constant region, dependingon the context. The origin of the starting Fc region or startingconstant region is not limited, and it can be obtained from any organismof nonhuman animals or from a human. Furthermore, the startingFcRn-binding domain can be obtained from cynomolgus monkeys, marmosets,Rhesus monkeys, chimpanzees, and humans. Starting Fc regions or startingconstant regions may be obtained from human IgG1, but are not limited toany particular IgG class. This means that an Fc region of human IgG1,IgG2, IgG3, or IgG4 can be used as an appropriate starting FcRn-bindingdomain, and an Fc region or constant region of an IgG class or subclassderived from any organism can be used as a starting Fc region or as astarting constant region. Examples of native IgG variants or modifiedforms are described in, for example, Strohl, Curr. Opin. Biotechnol.20(6):685-691 (2009); Presta, Curr. Opin. Immunol. 20(4):460-470 (2008);Davis et al., Protein Eng. Des. Sel. 23(4):195-202 (2010),WO2009/086320, WO2008/092117; WO2007/041635; and WO2006/105338).

Within the scope of Disclosures A and B described herein, amino acidresidues of the starting FcRn-binding domain, starting Fc region, orstarting constant region may contain, for example, one or moremutations: for example, substitution mutations with amino acid residuesthat are different from the amino acid residues in the starting Fcregion or starting constant region; insertions of one or more amino acidresidues into the amino acid residues in the starting Fc region orstarting constant region; or deletions of one or more amino acidresidues from the amino acid residues of the starting Fc region orstarting constant region. The amino acid sequences of Fc regions orconstant regions after modifications may be preferably amino acidsequences containing at least a portion of an Fc region or constantregion that does not occur naturally. Such variants necessarily have asequence identity or similarity of less than 100% to the starting Fcregions or starting constant regions. For example, the variants have anamino acid sequence identity or similarity of about 75% to less than100%, more preferably about 80% to less than 100%, even more preferablyabout 85% to less than 100%, still more preferably about 90% to lessthan 100%, and yet more preferably about 95% to less than 100% to theamino acid sequence of the starting Fc region or starting constantregion. In a non-limiting example, at least one amino acid is differentbetween a modified Fc region or constant region of Disclosure A or B andthe starting Fc region or starting constant region.

Within the scope of Disclosures A and B described herein, an Fc regionor constant region that has FcRn-binding activity in an acidic pH rangeand/or in a neutral pH range may be obtained by any method.Specifically, a variant of Fc region or constant region that hasFcRn-binding activity in an acidic pH range and/or in a neutral pH rangemay be obtained by modifying amino acids of a human IgG-type antibodywhich can be used as the starting Fc region or starting constant region.IgG-type antibody Fc regions or constant regions suitable formodification include, for example, the Fc regions or constant regions ofhuman IgG (IgG1, IgG2, IgG3, and IgG4, and variants thereof), andmutants spontaneously generated therefrom are also included in the IgGFc regions or constant regions. For the Fc regions or constant regionsof human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, anumber of allotype sequences due to genetic polymorphism are describedin “Sequences of proteins of immunological interest”, NIH PublicationNo. 91-3242, and any of them may be used in Disclosure A or B. Inparticular, for the human IgG1 sequence, the amino acid sequence ofpositions 356 to 358 according to EU numbering may be DEL or EEM.

In one embodiment of Disclosure A or B, the modification into otheramino acids is not particularly limited, as long as the resultingvariants have FcRn-binding activity in an acidic pH range and/or in aneutral pH range, and preferably in a neutral pH range. Sites of aminoacid modification to increase the FcRn-binding activity under a neutralpH condition are described, for example, in WO2013/046722. Suchmodification sites include, for example, one or more positions selectedfrom the group consisting of: position 221 to 225, 227, 228, 230, 232,233 to 241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289,291 to 312, 315 to 320, 324, 325, 327 to 339, 341, 343, 345, 360, 362,370, 375 to 378, 380, 382, 385 to 387, 389, 396, 414, 416, 423, 424, 426to 438, 440, and 442, according to EU numbering in the Fc region orconstant region of a human IgG antibody, as described in WO2013/046722.WO2013/046722 also describes, as a part of the preferred modificationsin the Fc region or constant region, for example, modification of one ormore amino acids selected from the group consisting of: the amino acidat position 256 to Pro, the amino acid at position 280 to Lys, the aminoacid at position 339 to Thr, the amino acid at position 385 to His, theamino acid at position 428 to Leu, and the amino acid at position 434 toTrp, Tyr, Phe, Ala, or His, according to EU numbering. The number ofamino acids to be modified is not particularly limited, and modificationmay be performed at a single position alone or at two or more positions.Modification of these amino acid residues can enhance the FcRn bindingof the Fc region or constant region of an IgG-type antibody under aneutral pH condition. Modification of these amino acid residues may alsobe introduced appropriately into antibodies of Disclosure A or B.

In a further or alternative embodiment, it is also possible to useappropriate amino acid modification sites for increasing theFcRn-binding activity under an acidic pH condition. Among suchmodification sites, one or more modification sites that allow anincrease in the FcRn binding also in a neutral pH range can beappropriately used in Disclosure A or B. Such modification sitesinclude, for example, those reported in WO2011/122011, WO2013/046722,WO2013/046704, and WO2013/046722. The sites of amino acids that allowsuch modification of the constant region or Fc region of a humanIgG-type antibody and the types of amino acids after modification arereported in Table 1 of WO2013/046722. WO2013/046722 also describes, asparticularly preferred, modification sites in the constant region or Fcregion, for example, the location of one or more amino acid positionsselected from the group consisting of position 237, 238, 239, 248, 250,252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305,307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380,382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436, according toEU numbering. Modification of these amino acid residue positions canalso enhance the human FcRn binding of the FcRn-binding domain in aneutral pH range. WO2013/046722 also describes, as a part of thepreferred modification in the IgG-type constant region or Fc region, forexample, modification of one or more amino acid residues selected fromthe group consisting of: (a) the amino acid at position 237 to Met; (b)the amino acid at position 238 to Ala; (c) the amino acid at position239 to Lys; (d) the amino acid at position 248 to Ile; (e) the aminoacid at position 250 to any one of Ala, Phe, Ile, Met, Gln, Ser, Val,Trp, and Tyr; (f) the amino acid at position 252 to any one of Phe, Trp,and Tyr; (g) the amino acid at position 254 to Thr; (h) the amino acidat position 255 to Glu; (i) the amino acid at position 256 to any one ofAsp, Glu, and Gln; (j) the amino acid at position 257 to any one of Ala,Gly, Ile, Leu, Met, Asn, Ser, Thr, and Val; (k) the amino acid atposition 258 to His; (l) the amino acid at position 265 to Ala; (m) theamino acid at position 270 to Phe; (n) the amino acid at position 286 toeither Ala or Glu; (o) the amino acid at position 289 to His; (p) theamino acid at position 297 to Ala; (q) the amino acid at position 298 toGly; (r) the amino acid at position 303 to Ala; (s) the amino acid atposition 305 to Ala; (t) the amino acid at position 307 to any one ofAla, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Val, Trp, and Tyr; (u) the amino acid at position 308 to any one of Ala,Phe, Ile, Leu, Met, Pro, Gln, and Thr; (v) the amino acid at position309 to any one of Ala, Asp, Glu, Pro, and Arg; (w) the amino acid atposition 311 to any one of Ala, His, and Ile; (x) the amino acid atposition 312 to either Ala or His; (y) the amino acid at position 314 toeither Lys or Arg; (z) the amino acid at position 315 to either Ala orHis; (aa) the amino acid at position 317 to Ala; (ab) the amino acid atposition 325 to Gly; (ac) the amino acid at position 332 to Val; (ad)the amino acid at position 334 to Leu; (ae) the amino acid at position360 to His; (af) the amino acid at position 376 to Ala; (ag) the aminoacid at position 380 to Ala; (ah) the amino acid at position 382 to Ala;(ai) the amino acid at position 384 to Ala; (aj) the amino acid atposition 385 to either Asp or His; (ak) the amino acid at position 386to Pro; (al) the amino acid at position 387 to Glu; (am) the amino acidat position 389 to either Ala or Ser; (an) the amino acid at position424 to Ala; (ao) the amino acid at position 428 to any one of Ala, Asp,Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, andTyr; (ap) the amino acid at position 433 to Lys; (aq) the amino acid atposition 434 to Ala, Phe, His, Ser, Trp, and Tyr; and (ar) the aminoacid at position 436 to His; according to EU numbering. The number ofamino acids to be modified is not particularly limited, and modificationmay be performed at a single position alone or at two or more positions.Combinations of amino acid modifications at two or more positionsinclude, for example, those shown in Table 2 of WO2013/046722.Modification of these amino acid residues may also be appropriatelyintroduced into antibodies of Disclosures A and B.

In one embodiment, the FcRn-binding activity of the FcRn-binding domainof an antibody of Disclosure A or B has been increased when compared tothat of a reference antibody containing an Fc region or constant regionof a native IgG or that of a reference antibody containing a starting Fcregion or starting constant region. Namely, the FcRn-binding activity ofan Fc region variant or constant region variant of Disclosure A or B, oran antibody containing such variant is greater than that of thereference antibody). This can mean that when compared to theFcRn-binding activity of the reference antibody, that of an antibody ofDisclosure A or B can be, for example: 55% or greater, 60% or greater,65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% orgreater, 90% or greater, 95% or greater, 100% or greater, 105% orgreater, preferably 110% or greater, 115% or greater, 120% or greater,125% or greater, more preferably 130% or greater, 135% or greater, 140%or greater, 145% or greater, 150% or greater, 155% or greater, 160% orgreater, 165% or greater, 170% or greater, 175% or greater, 180% orgreater, 185% or greater, 190% or greater, 195% or greater, 2-fold orgreater, 2.5-fold or greater, 3-fold or greater, 3.5-fold or greater,4-fold or greater, 4.5-fold or greater, or 5-fold or greater.

In one embodiment, amino acid sequences to be modified in an antibody ofDisclosure A or B can preferably contain human sequences (sequencesfound in native human-derived antibodies) in order to not increase theimmunogenicity of the antibody when the antibody is administered in vivo(preferably, into a human body). Alternatively, after modification,mutations may be introduced at positions other than the sites of aminoacid modification in such a way that one or more of the FRs (FR1, FR2,FR3, and FR4) is substituted with a human sequence. Methods forsubstituting FR(s) with a human sequence are known in the art andinclude, but are not limited to that reported in Ono et al., Mol.Immunol. 36(6):387-395 (1999). Humanization methods are known in the artand include, but are not limited to that reported in, Methods36(1):43-60 (2005).

In one embodiment, the framework region sequences (also referred to as“FR sequences”) of the heavy chain and/or light chain variable region ofan antibody of Disclosure A or B may contain human germ-line frameworksequences. When the framework sequences are completely human germ-linesequences, the antibody is expected to induce little or no immunogenicreaction when administered to humans (for example, to treat or prevent acertain disease).

FR sequences preferably can include, for example, fully human FRsequences such as those shown in V-Base (vbase.mrc-cpe.cam.ac.uk/).These FR sequences can be appropriately used for Disclosure A or B. Thegerm-line sequences may be categorized based on their similarity(Tomlinson et al. (J. Mol. Biol. 227:776-798 (1992); Williams et al.(Eur. J. Immunol. 23:1456-1461 (1993); and Cox et al. (Nat. Genetics7:162-168 (1994)). Preferred germ-line sequences can be appropriatelyselected from Vκ, which is categorized into seven subgroups; Vλ, whichis categorized into ten subgroups; and VH, which is categorized intoseven subgroups.

Fully human VH sequences can preferably include, for example, VHsequences of: subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1-18,VH1-24, VH1-45, VH1-46, VH1-58, and VH1-69); subgroup VH2 (for example,VH2-5, VH2-26, and VH2-70); subgroup VH3 (VH3-7, VH3-9, VH3-11, VH3-13,VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38,VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, andVH3-74); subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59,and VH4-61); subgroup VH5 (VH5-51); subgroup VH6 (VH6-1); or subgroupVH7 (VH7-4 and VH7-81). These are also described in, for example,Matsuda et al. (I Exp. Med. 188:1973-1975 (1998)), and those of ordinaryskill in the art can appropriately design antibodies based oninformation of these sequences. It can be also preferable to use otherfully human FR sequence or sequences of regions that are equivalentthereto.

Fully human Vκ sequences can preferably include, for example: A20, A30,L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2, O4,O8, O12, O14, or O18, which are classified as subgroup Vk1; A1, A2, A3,A5, A7, A17, A18, A19, A23, O1, and O11, which are classified assubgroup Vk2; A11, A27, L2, L6, L10, L16, L20, and L25, which areclassified as subgroup Vk3; B3, classified as subgroup Vk4; B2 (alsoreferred to as “Vk5-2”), classified as subgroup Vk5; or A10, A14, andA26, which are classified as subgroup Vk6 (Kawasaki et al. (Eur. J.Immunol. 31:1017-1028 (2001)); (Hoppe Seyler Biol. Chem. 374:1001-1022(1993)); Brensing-Kuppers et al. (Gene 191:173-181 (1997)).

Fully human Vλ sequences can preferably include, for example: V1-2,V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18, V1-19,V1-20, and V1-22, which are classified as subgroup VL1; V2-1, V2-6,V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19, which areclassified as subgroup VL2; V3-2, V3-3, and V3-4, which are classifiedas subgroup VL3; V4-1, V4-2, V4-3, V4-4, and V4-6, which are classifiedas subgroup VL4; or V5-1, V5-2, V5-4, and V5-6, which are classified assubgroup VL5 (Kawasaki et al. Genome Res. 7:250-261 (1997)).

Normally, these FR sequences are different from one another at one ormore amino acid residues. These FR sequences can be used in themodification of antibody amino acid residues. Fully human FR sequencesthat may be used in the modification also include, for example, KOL,NEWM, REI, EU, TUR, TEI, LAY, and POM (see, for example, aforementionedKabat et al. (1991); Wu et al. (J. Exp. Med. 132:211-250 (1970)).

Within the scope of Disclosures A and B described herein, “flexibleresidues” can refer to amino acid residue variations that are present atpositions showing high amino acid diversity at which the light chain orheavy chain variable regions have several different amino acids when theamino acid sequences of known and/or native antibodies orantigen-binding domains are compared. Positions showing high diversityare generally located in the CDRs. The data provided by Kabat, Sequencesof Proteins of Immunological Interest (National Institute of HealthBethesda Md.) (1987 and 1991), can be effective in determining suchpositions with high diversity in known and/or native antibodies.Furthermore, several databases on the Internet(vbase.mrc-cpe.cam.ac.uk/, bioinf.org.uk/abs/index.html) provide acollection of numerous human light chain and heavy chain sequences andtheir locations. Information on these sequences and locations is usefulto determine the locations of flexible residues. Without limitations,for example, when an amino acid residue at a particular position has avariability of, preferably, 2 to 20, 3 to 19, 4 to 18, 5 to 17, 6 to 16,7 to 15, 8 to 14, 9 to 13, or 10 to 12 amino acid residues, the positioncan be judged to show (high) diversity.

In an embodiment, it can be understood that where an antibody ofDisclosure A or B contains the whole or a portion of the light chainvariable region and/or heavy chain variable region, the antibody maycontain one or more appropriate flexible residues, if needed. Forexample, a heavy chain and/or light chain variable region sequenceselected to have an FR sequence which originally contains amino acidresidues that change the antigen-binding activity of an antibodyaccording to the ion concentration (hydrogen ion concentration orcalcium ion concentration) conditions can be designed to contain, otheramino acid residues in addition to these amino acid residues. In thiscase, for example, the number and locations of the flexible residues canalso be determined without being limited to a specific embodiment, aslong as the antigen-binding activity of the antibody of Disclosure A orB changes according to the ion concentration condition. Specifically,the CDR sequence and/or FR sequence of a heavy chain and/or light chainmay contain at least one flexible residue. For example, where the ionconcentration is calcium ion concentration, flexible residues that canbe introduced into the light-chain variable region sequence(aforementioned Vk5-2) include, but are not limited to, one or moreamino acid residue positions shown in Table 1 or Table 2. Likewise,appropriate flexible residues can be introduced, for example, into anion concentration-dependent antibody or antibody without such ionconcentration dependency, containing the whole or a portion of the lightchain variable region and/or heavy chain variable region, in which atleast one amino acid residue that may be exposed on the antibody surfacehas been modified such that the pI is increased.

TABLE 1 Kabat CDR numbering Amino acid in 70% of the total CDR1 28 S:100% 29 I: 100% 30 E: 72% N: 14% S: 14% 31 D: 100% 32 D: 100% 33 L: 100%34 A: 70% N: 30% CDR2 50 E: 100% 51 A: 100% 52 S: 100% 53 H: 5% N: 25%S: 45% T: 25% 54 L: 100% 55 Q: 100% 56 S: 100% CDR3 90 Q: 100% 91 H: 25%S: 15% R: 15% Y: 45% 92 D: 80% N: 10% S: 10% 93 D: 5% G: 10% N: 25% S:50% R: 10% 94 S: 50% Y: 50% 95 P: 100% 96 L: 50% Y: 50% (Positions areshown according to Kabat numbering.)

TABLE 2 Kabat CDR numbering Amino acid in 30% of the total CDR1 28 S:100% 29 I: 100% 30 E: 83% S: 17% 31 D: 100% 32 D: 100% 33 L: 100% 34 A:70% N: 30% CDR2 50 H: 100% 51 A: 100% 52 S: 100% 53 H: 5% N: 25% S: 45%T: 25% 54 L: 100% 55 Q: 100% 56 S: 100% CDR3 90 Q: 100% 91 H: 25% S: 15%R: 15% Y: 45% 92 D: 80% N: 10% S: 10% 93 D: 5% G: 10% N: 25% S: 50% R:10% 94 S: 50% Y: 50% 95 P: 100% 96 L: 50% Y: 50% (Positions are shownaccording to Kabat numbering.)

In one embodiment, when humanizing a chimeric antibody, the pI of thechimeric antibody is increased by modifying one or more amino acidresidues that can be exposed on the antibody surface as to produce ahumanized antibody of Disclosure A or B with a shortened plasmahalf-life as compared to the chimeric antibody absent such modification.The modification of amino acid residues that can be exposed on thesurface of the humanized antibody can be carried out before orconcurrently with humanization of the antibody. Alternatively, by usingthe humanized antibody as a starting material, amino acid residues thatcan be exposed on the surface may be modified to further alter the pI ofthe humanized antibody.

Adams et al (Cancer Immunol. Immunother. 55(6):717-727 (2006)) reportsthat the humanized antibodies, trastuzumab (antigen: HER2), bevacizurnab(antigen: VEGF), and pertuzumab (antigen: HER2), which were humanizedusing the same human antibody FR sequences, were almost comparable inplasma pharmacokinetics. Specifically, it can be understood that theplasma pharmacokinetics is almost comparable when humanization isperformed using the same FR sequences. According to one embodiment ofDisclosure A, the antigen concentration in plasma is reduced byincreasing the antibody's pI by modifying amino acid residues that canbe exposed on the antibody surface, in addition to the humanizationstep. In an alternative embodiment for Disclosure A or B, humanantibodies can be used. By modifying amino acid residues that can beexposed on the surface of a human antibody produced from a humanantibody library, a human antibody-producing mouse, a recombinant cell,etc., and increasing the pI of the human antibody, the ability of theoriginally-produced human antibody to eliminate antigen from plasma canbe increased.

In one embodiment, antibodies of Disclosure A may contain modified sugarchains. Antibodies with modified sugar chains include, for example,antibodies with modified glycosylation (WO99/54342), antibodies thatlack fucose (WO00/61739; WO02/31140, WO2006/067847; WO2006/067913), andantibodies having sugar chains with bisecting GlcNAc (WO02/79255).

In one embodiment, antibodies of Disclosure A or B can be used, forexample, in techniques for exhibiting increased antitumor activitiesagainst cancer cells or in techniques for promoting elimination ofantigens that are harmful to the organism from the plasma.

In an alternative embodiment, Disclosure A or B relate to libraries ofthe ion concentration-dependent antigen-binding domains with anincreased pI or ion concentration-dependent antibodies with an increasedpI, as described above.

In an alternative embodiment, Disclosure A or B relates to nucleic acids(polynucleotides) encoding the above-described ionconcentration-dependent antigen-binding domains with an increased pI orion concentration-dependent antibodies with an increased pI. In aspecific embodiment, the nucleic acids can be obtained using appropriateknown methods. For specific embodiments, for example, WO2009/125825,WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704,WO2000/042072, WO2006/019447, WO2012/115241, WO2013/047752,WO2013/125667, WO2014/030728, WO2014/163101, WO2013/081143,WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, andWO2012/093704 can be referred to, each of these are incorporated hereinby reference in their entirety.

In one embodiment, nucleic acids of Disclosure A or B can be isolated orpurified nucleic acids. Nucleic acids encoding the antibodies ofDisclosure A or B may be any genes, and may be DNA or RNA, or othernucleic acid analogs.

Within Disclosures A and B described herein, when amino acids of anantibody are modified, the amino acid sequence of the antibody beforemodification may be a known sequence or the amino acid sequence of anantibody newly obtained. For example, antibodies can be obtained fromantibody libraries, or by cloning nucleic acids encoding the antibodyfrom hybridomas or B cells that produce monoclonal antibodies. Themethods for obtaining nucleic acids encoding an antibody from hybridomasmay use the techniques of: performing immunization by conventionalimmunization methods using an antigen of interest or cells expressingthe antigen of interest as a sensitizing antigen; fusing the resultingimmune cells with known parental cells by conventional cell fusionmethods; screening for monoclonal antibody-producing cells (hybridomas)by conventional screening methods; synthesizing cDNAs of the variableregion (V region) of the antibody using reverse transcriptase from mRNAsof the obtained hybridomas; and linking the cDNA to a DNA encoding anantibody constant region (C region) of interest.

Sensitizing antigens which are used to obtain nucleic acids encoding theabove-described heavy chain and light chain include, but are not limitedto, both complete antigens with immunogenicity and incomplete antigensincluding haptens which exhibit no immunogenicity. For example, it ispossible to use whole proteins of interest or partial peptides of theproteins. In addition, substances that are composed of polysaccharides,nucleic acids, lipids, and other compositions known to be potentialantigens. Thus, in some embodiments, antigens for the antibodies ofDisclosure A or B are not particularly limited. The antigens can beprepared by, for example, baculovirus-based methods (see, e.g.,WO98/46777). Hybridomas can be produced, for example, according to themethod of G. Kohler and C. Milstein, Methods Enzymol. 73:3-46 (1981)).When the immunogenicity of an antigen is low, immunization may beperformed by linking the antigen with a macromolecule havingimmunogenicity, such as albumin. Alternatively, if necessary, solubleantigens can be prepared by linking the antigen with other molecules.When a transmembrane molecule such as membrane antigens (for example,receptors) is used as an antigen, a portion of the extracellular regionof the membrane antigen can be used as a fragment, or cells expressingthe transmembrane molecule on their surface may be used as an immunogen.

In some embodiments, antibody-producing cells can be obtained byimmunizing an animal with an appropriate sensitizing antigen describedabove. Alternatively, antibody-producing cells can be prepared by invitro immunization of lymphocytes that are capable of producingantibodies. Various mammals can be used for immunization and otherroutine antibody producing procedures. Commonly used animals includerodents, lagomorphs, and primates. The animals may include, for example,rodents such as mice, rats, and hamsters; lagomorphs such as rabbits;and primates including monkeys such as cynomolgus monkeys, rhesusmonkeys, baboons, and chimpanzees. In addition, transgenic animalscarrying a human antibody gene repertoire are also known, and theseanimals can be used to obtain human antibodies (see, e.g., WO96/34096;Mendez et al., Nat. Genet. 15:146-156 (1997); WO93/12227, WO92/03918,WO94/02602, WO96/34096, and WO96/33735). Instead of using suchtransgenic animals, it is also possible to obtain desired humanantibodies having antigen-binding activity by, for example, sensitizinghuman lymphocytes in vitro with desired antigens or cells expressing thedesired antigens and then fusing the sensitized lymphocytes with humanmyeloma cells such as U266 (JP Pat. Publ. No. H01-59878).

Animal immunization can be carried out, for example, by appropriatelydiluting and suspending a sensitizing antigen in phosphate bufferedsaline (PBS), physiological saline, or others, and mixing it with anadjuvant to emulsify, if needed; and then injecting it intraperitoneallyor subcutaneously into animals. Then, the sensitizing antigen mixed withFreund's incomplete adjuvant can be preferably administered severaltimes every four to 21 days. Antibody production can be confirmed, forexample, by measuring the titer of the antibody of interest in animalsera.

Antibody-producing cells obtained from lymphocytes or animals immunizedwith a desired antigen can be fused with myeloma cells to generatehybridomas using conventional fusing agents (for example, polyethyleneglycol) (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, 1986, 59-103). If needed, hybridomas are cultured andexpanded, and the binding specificity of antibodies produced by thehybridomas is assessed by, for instance, immunoprecipitation,radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).Then, if needed, antibody-producing hybridomas whose specificity,affinity, or activity of interest has been determined may also besubcloned by methods such as limiting dilution.

Nucleic acids encoding the selected antibody can be cloned fromhybridomas or antibody-producing cells (sensitized lymphocytes, etc.)using probes that can specifically bind to the antibody (for example,oligonucleotides complementary to sequences encoding the antibodyconstant regions). Alternatively, the nucleic acids can be cloned frommRNA using RT-PCR. Heavy chains and light chains for use in producingantibodies of Disclosure A or B may be derived from antibodies that, forexample, belong to any of Ig antibody classes and subclasses, and IgGmay be preferred.

In one embodiment, nucleic acids encoding amino acid sequences thatconstitute the heavy chain (the whole or a portion thereof) and/or lightchain (the whole or a portion thereof) of an antibody of Disclosure A orB, for example, are modified by genetic engineering techniques.Recombinant antibodies with artificial sequence modification to, forexample, reduce heterologous antigenicity against humans, such aschimeric antibodies or humanized antibodies, may be appropriatelygenerated by, for example, modifying nucleotide residues encoding aminoacid sequences associated with components of antibodies such as mouseantibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheepantibodies, or camel antibodies. Chimeric antibodies can be obtained,for example, by ligating a DNA encoding a mouse-derived antibodyvariable region with a DNA encoding a human antibody constant region andincorporating the ligated DNA coding sequence into an expression vector,then introducing the resulting recombinant vector into a host to expressthe genes. Humanized antibodies, which are also referred to as reshapedhuman antibodies, are antibodies in which human antibody FR(s) arelinked in frame with antibody CDR(s) isolated from non-human mammals,such as mice, to form a coding sequence. A DNA sequence encoding such ahumanized antibody can be synthesized by overlap extension PCR using anumber of oligonucleotides as templates. Materials and experimentalmethods for overlap extension PCR are described in WO98/13388 andothers. For example, a DNA encoding the amino acid sequence of, forexample, an antibody variable region of Disclosure A or B may beobtained by overlap extension PCR using a number of oligonucleotidesdesigned to have overlapping nucleotide sequences. The overlapping DNAis then linked in frame to a DNA encoding a constant region to form acoding sequence. The DNA linked as described above may be then insertedinto an expression vector so that the DNA can be expressed, and theresulting vector may be introduced into a host or host cell. Theantibody encoded by the DNA can be expressed by raising the host orculturing the host cells. The expressed antibody can be appropriatelypurified from culture media of the host or others (EP239400;WO96/02576). Furthermore, the FR(s) of a humanized antibody which arelinked via CDR(s) may be selected, for example, to allow the CDRs toform an antigen-binding site suitable for the antigen. If necessary,amino acid residues that constitute FR(s) of a variable region of theselected antibody, for example, can be modified with appropriatesubstitution.

In one embodiment, to express antibodies of Disclosure A or B orfragments thereof, nucleic acid cassettes may be cloned into appropriatevectors. For such purposes, several types of vectors, such as phagemidvectors are available. In general, phagemid vectors can contain variouselements including regulatory sequences such as promoters or signalsequences, phenotype selection genes, replication origins, and othernecessary elements.

Methods for introducing desired amino acid modifications into antibodieshave been established in the field of the art. For example, librariescan be constructed by introducing at least one modified amino acidresidue that can be exposed on the surface of antibodies of Disclosure Aor B and/or at least one amino acid that can change the antigen-bindingactivity of antibodies according to the ion concentration condition.Additionally, if needed, flexible residues can be added using the methodof Kunkel et al. (Methods Enzymol. 154:367-382 (1987)).

In an alternative embodiment, Disclosure A relates to vectors containingnucleic acids encoding an above-described ion concentration-dependentantigen-binding domain with increased pI or an above-described ionconcentration-dependent antibody with increased pI. In a specificembodiment, the vectors can be obtained by, for example, vectors asdescribed in WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722,WO2013/046704, WO2000/042072, WO2006/019447, WO2012/115241,WO2013/047752, WO2013/125667, WO2014/030728, WO2014/163101,WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159,WO2012/016227, or WO2012/093704, each of which is incorporated herein byreference in their entirety.

In one embodiment, the nucleic acids encoding embodiments of DisclosureA or B may be operably cloned (inserted) into appropriate vectors andintroduced into host cells. For example, when E. coli is used as a host,vectors include the cloning vector, pBluescript vector (Stratagene) orany of various other commercially available vectors.

In one embodiment, expression vectors are useful as vectors containing anucleic acid for Disclosure A or B. Expression vectors can be used toallow polypeptide expression in vitro, in E. coli, in culture cells, orin vivo. For example, it is possible to use pBEST vector (Promega) forin vitro expression; pET vector (Invitrogen) for E. coli expression;pME18S-FL3 vector (GenBank Accession No. AB009864) for culture cellexpression; and pME18S vector (Takebe et al., Mol. Cell Biol. 8:466-472(1988)) for in vivo expression. DNAs can be inserted into vectors byconventional methods, for example, by ligase reaction using restrictionenzyme sites (see, Current protocols in Molecular Biology edit. Ausubelet al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

In an alternative embodiment, Disclosure A relates to a host or hostcells that comprise a vector containing a nucleic acid encoding anabove-described ion concentration-dependent antigen-binding domain withincreased pI or an above-described ion concentration-dependent antibodywith increased pI. In a specific embodiment, the host or host cells canbe prepared by, for example, methods described in WO2009/125825,WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704,WO2000/042072, WO2006/019447, WO2012/115241, WO2013/047752,WO2013/125667, WO2014/030728, WO2014/163101, WO2013/081143,WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, orWO2012/093704, each of which is incorporated herein by reference intheir entirety.

The type of host cell of Disclosure A or B is not particularly limited,and the host cells include, for example, bacterial cells such as E.coli, as well as various animal cells. The host cells can beappropriately used as production systems for producing and expressingthe antibodies. Both eukaryotic and prokaryotic cells can be used.

Eukaryotic cells for use as host cells include, for example, animalcells, plant cells, and fungal cells. Examples of animal cells includemammalian cells, for example, CHO (Puck et al., J. Exp. Med. 108:945-956(1995)), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, andVero; amphibian cells, for example, Xenopus oocyte (Valle et al., Nature291:338-340 (1981)); and insect cells, for example, Sf9, Sf21, and Tn5.Recombinant vectors or others can be introduced into host cells, forexample, using calcium phosphate methods, DEAE-dextran methods, methodsusing cationic liposome DOTAP (Boehringer-Mannheim), electroporation,and lipofection.

Plant cells that are known to serve as a protein production systeminclude, for example, Nicotiana tabacum-derived cells and duckweed(Lemna minor)-derived cells. Calluses can be cultured from these cellsto produce antibodies of Disclosure A or B. Fungal cell-based proteinproduction systems include those using yeast cells, for example, cellsof genus Saccharomyces such as Saccharomyces cerevisiae andSchizosaccharomyces pombe; and cells of filamentous fungi, for example,genus Aspergillus such as Aspergillus niger. When prokaryotic cells areused, bacterial cell-based production systems can be used. Bacterialcell-based production systems include, for example, those using Bacillussubtilis as well as E. coli.

To produce an antibody of Disclosure A or B using host cells, the hostcells are transformed with an expression vector containing a nucleicacid encoding an antibody of Disclosure A or B and cultured to expressthe nucleic acid. For example, when animal cells are used as a host,culture media may include, for example, DMEM, MEM, RPMI1640, and IMDM,which may be appropriately used in combination with serum supplementssuch as FBS or fetal calf serum (FCS). Alternatively, the cells may becultured serum free.

On the other hand, animals or plants can be used for in vivo productionsystems for producing antibodies of Disclosure A or B, For example, anucleic acid(s) encoding an antibody of Disclosure A or B can beintroduced into such animals or plants to produce the antibody in vivo,and the antibody can then be collected from the animals or plants.

When animals are used as a host, production systems using mammals orinsects are available. Preferred mammals include, but are not limitedto, goats, pigs, sheep, mice, and bovines (Vicki Glaser, SPECTRUMBiotechnology Applications (1993)). Transgenic animals can also be used.

In one example, a nucleic acid encoding an antibody of Disclosure A or Bis prepared as a fusion gene with a gene encoding a polypeptide that isspecifically included in milk, such as goat β-casein. Then, goat embryosare injected with a polynucleotide fragment containing the fusion geneand transplanted into a female goat. The antibody of interest can beobtained from milk produced by the transgenic goats, which are born fromgoats that received the embryos, or from their offspring. Hormones canbe appropriately administered to the transgenic goats to increase thevolume of milk containing the antibody produced by the goats (Ebert etal., Bio/Technology 12:699-702 (1994)).

Insects for use in producing antibodies of Disclosure A or B include,for example, silkworms. When silkworms are used, baculoviruses whoseviral genome is inserted with a polynucleotide encoding an antibody ofinterest is used to infect the silkworm. The antibody of interest can beobtained from the body fluids of the infected silkworms (Susumu et al.,Nature 315:592-594 (1985)).

When plants are used for producing antibodies of Disclosure A or B,tobacco may be used. When tobacco is used, a recombinant vectorresulting from insertion of a polynucleotide encoding an antibody ofinterest into a plant expression vector, for example, pMON 530 may beintroduced into bacteria such as Agrobacterium tumefaciens. Theresulting bacteria can be used to infect tobacco, for example, Nicotianatabacum (Ma et al., Eur. J. Immunol. 24:131-138 (1994)) and the desiredantibody is obtained from the leaves of the infected tobacco. Suchmodified bacteria can be also used to infect duckweed (Lemna minor), andthe desired antibody is obtained from cloned cells of the infectedduckweed (Cox et al. Nat. Biotechnol. 24(12):1591-1597 (2006)).

In order to secrete the antibody which is expressed in the host cellsinto the lumen of the endoplasmic reticulum, into the periplasmic space,or into the extracellular environment, suitable secretion signals may beincorporated into the polypeptide of interest. Such signals may beendogenous to the antibody of interest or may be a heterogeneous signalknown in the art.

The antibody of Disclosure A or B produced as described above may beisolated from the inside or outside (such as media and milk) of hostcells or a host, and purified to a substantially pure and homogenousantibody. The antibodies can be suitably isolated and purified, forexample, by appropriately selecting and combining chromatographiccolumns, filtration, ultrafiltration, salting out, solventprecipitation, solvent extraction, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis,recrystallization, and others. Chromatography includes, for example,affinity chromatography, ion exchange chromatography, hydrophobicchromatography, gel filtration chromatography, reverse phasechromatography, and adsorption chromatography. Such chromatography canbe performed, for example, by using liquid chromatography such as HPLCand FPLC. Columns for use in affinity chromatography may be Protein Acolumn or Protein G column. Protein A column include, for example, HyperD, POROS, Sepharose F.F. (Pharmacia).

The antibody can be modified or the peptide can be partially deleted bytreating the antibody with appropriate protein modifying enzymes beforeor after antibody purification, as necessary. For such protein modifyingenzymes, for example, trypsin, chymotrypsin, lysyl endopeptidase,protein kinases, and glucosidases can be used.

In an alternative embodiment, Disclosure A relates to methods forproducing antibodies containing an antigen-binding domain whoseantigen-binding activity changes according to the ion concentrationcondition, which may comprise culturing the host cells or raising thehosts and collecting antibodies from cultures of these cells, materialssecreted from the hosts, or by other means known in the art.

In one embodiment, Disclosure A relates to a production method whichcomprises any one or more steps selected from the group consisting of:(a) selecting an antibody which can promote elimination of an antigenfrom plasma; (b) selecting an antibody with enhanced binding activity toan extracellular matrix; (c) selecting an antibody with enhancedFcγR-binding activity under a neutral pH condition; (d) selecting anantibody with enhanced FcγRIIb-binding activity under a neutral pHcondition; (e) selecting an antibody with maintained or enhancedFcγRIIb-binding activity and decreased binding activity to one or moreactivating FcγR selected from the group consisting of FcγRIa, FcγRIb,FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa; (f) selecting an antibody withenhanced FcRn-binding activity under a neutral pH condition; (g)selecting an antibody with a pI; (h) confirming the pI of the collectedantibody, and then selecting an antibody with an increased pI; and (i)selecting an antibody whose antigen-binding activity is changed orincreased according to ion concentration conditions, as compared to areference antibody.

Here, the reference antibody includes, but is not limited to, a nativeantibody (for example, a native Ig antibody, preferably a native IgGantibody) and an antibody before modification (an antibody prior to orduring library construction, for example, an ion concentration-dependentantibody prior to increasing its pI, or an antibody with increased pIprior to conferring an ion concentration-dependent antigen-bindingdomain).

After producing antibodies of Disclosure A, the resulting antibodies maybe assessed by antibody pharmacokinetic assay using plasma such as ofmice, rats, rabbits, dogs, monkeys, humans, to select antibodies withenhanced antigen elimination from plasma as compared to the referenceantibody.

Alternatively, after producing antibodies of Disclosure A, the resultingantibodies may be compared with a reference antibody in terms of theextracellular matrix-binding ability by electrochemiluminescence orothers to select antibodies with increased binding to extracellularmatrix.

Alternatively, after producing antibodies of Disclosure A, the resultingantibodies may be compared with a reference antibody in terms of thebinding activity to various FcγRs under a neutral pH condition usingBIACORE® or others to select antibodies with increased binding activityto various FcγRs under the neutral pH condition. In this case, thevarious FcγRs may be a type of FcγR of interest, for example, FcγRIIb.Similarly, it is also possible to select antibodies whoseFcγRIIb-binding activity (under a neutral pH condition) has beenmaintained or increased and their binding activity to one or moreactivating FcγR selected from the group consisting of FcγRIa, FcγRIb,FcγRIc, FcγRIIIb and FcγRIIa, and so on, has been reduced. In suchcases, FcγR can be FcγR.

Alternatively, after producing antibodies of Disclosure A, the resultingantibodies may be compared with a reference antibody in terms of theFcRn-binding activity under a neutral pH condition using BIACORE orother known techniques to select antibodies with increased FcRn-bindingactivity under the neutral pH condition. In this case, the FcRn can behuman FcRn.

Alternatively, after producing antibodies of Disclosure A, the resultingantibodies may be evaluated for their pI by isoelectric focusing orothers to select antibodies with increased pI as compared to thereference antibody. In this case, it is possible to select antibodieswhose pI value has been increased, for example, by at least 0.01, 0.03,0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 or more, or at least 0.6, 0.7, 0.8, or0.9 or more; or antibodies whose pI value has been increased by at least1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 or more, or at least 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 or more, or 3.0 or more.

Alternatively, after producing antibodies of Disclosure A, the resultingantibodies may be compared with a reference antibody in terms of bindingactivity to a desired antigen under low and high ion concentrationconditions using BIACORE or others to select antibodies whoseantigen-binding activity have been changed or increased according to theion concentration condition. The ion concentration may be, for example,hydrogen ion concentration or metal ion concentration. When the ionconcentration is a metal ion concentration, it can be, for example,calcium ion concentration. Whether the binding activity has been changedor increased may be assessed based on the presence of, for example: (a)an altered or enhanced antigen uptake by cells; (b) an altered orincreased ability to bind to different antigen molecules multiple times;(c) an altered or enhanced reduction of antigen concentration in plasma;or (d) an altered plasma retention of the antibody. Alternatively, anytwo or more of these selection methods may be appropriately combined, ifneeded.

In an alternative embodiment, Disclosure A relates to methods forproducing or screening for antibodies that contain an antigen-bindingdomain whose antigen-binding activity changes according to the ionconcentration condition and whose pI has been increased by modifying atleast one amino acid residue that can be exposed on the antibody surface(“ion concentration-dependent antibodies with increased pI”). Theproduction methods can be performed, for example, by appropriatelycombining as needed, the related embodiments described within the scopeof Disclosure A herein, for example, the embodiment of methods forproducing or screening for antibodies with increased pI described above,as well as the embodiment of methods for producing or screening forcalcium ion concentration-dependent antigen-binding domains or calciumion concentration-dependent antibodies whose antigen-binding activity ishigher under a high calcium ion concentration condition than under a lowcalcium ion concentration condition, or libraries thereof describedabove and/or the embodiment of methods for producing or screening forpH-dependent antigen-binding domains or pH-dependent antibodies whoseantigen-binding activity is higher in a neutral pH condition than in anacidic pH condition, or libraries thereof described above.

In an alternative embodiment, Disclosure A provides be a method forproducing or screening for an antibody containing an antigen-bindingdomain whose extracellular matrix-binding activity has been increased,wherein its antigen-binding activity changes according to the ionconcentration condition and its pI has been increased by modifying atleast one amino acid residue that can be exposed on the antibody surface(“ion concentration-dependent antibodies with increased pI”). Increasein pI of an ion concentration-dependent antibody may be contemplated inthe method. Such method can be performed, for example, by appropriatelycombining as needed, related embodiments described within the scope ofDisclosure A herein, for example, the embodiment of method for producingor screening for antibodies with an increased pI described above, aswell as the embodiment of methods for producing or screening for calciumion concentration-dependent antigen-binding domains or calcium ionconcentration-dependent antibodies whose antigen-binding activity ishigher under a high calcium ion concentration condition than under a lowcalcium ion concentration condition, or libraries thereof describedabove and/or the embodiment of methods for producing or screening forpH-dependent antigen-binding domains or pH-dependent antibodies whoseantigen-binding activity is higher under a neutral pH condition thanunder an acidic pH condition, or libraries thereof described above. Forexample, the resulting antibodies may be compared with a referenceantibody in terms of the extracellular matrix-binding ability byelectrochemiluminescence or other known techniques to select antibodieswith increased extracellular matrix binding.

Here, the reference antibody may include, but is not limited to, anative antibody (for example, a native Ig antibody, preferably a nativeIgG antibody) and an antibody before modification (an antibody prior toor during library construction, for example, an ionconcentration-dependent antibody before its pI is increased or anantibody with increased pI before it is conferred with an ionconcentration-dependent antigen-binding domain).

In an alternative embodiment, Disclosure A relates to a method forproducing an antibody comprising an antigen-binding domain whoseantigen-binding activity changes according to ion concentrationconditions, wherein the method comprises modifying at least one aminoacid residue that may be exposed on the surface of the antibody so as toincrease the isoelectric point (pI). In some embodiments, the amino acidresidue modification comprises a modification selected from the groupconsisting of: (a) substitution of a negatively charged amino acidresidue with an uncharged amino acid residue; (b) substitution of anegatively charged amino acid residue with a positively charged aminoacid residue; and (c) substitution of an uncharged amino acid residuewith a positively charged amino acid residue. In some embodiments, atleast one modified amino acid residue is substituted with histidine. Infurther embodiments, the antibody comprises a variable region and/or aconstant region, and an amino acid residue is modified in the variableregion and/or the constant region. In further embodiments, at least oneamino acid residue modified according to the method is in a position ina CDR or FR selected from the group consisting of: (a) position 1, 3, 5,8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68,71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110,and 112 in a FR of the heavy chain variable region; (b) position 31, 61,62, 63, 64, 65, and 97 in a CDR of the heavy chain variable region; (c)position 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42,43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81,85, 100, 103, 105, 106, 107, and 108 in a FR of the light chain variableregion; and (d) position 24, 25, 26, 27, 52, 53, 54, 55, and 56 in a CDRof the light chain variable region, according to Kabat numbering. In yetfurther embodiments, at least one amino acid residue modified accordingto the method is in a position in a CDR or FR selected from the groupconsisting of (a) position 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43,44, 77, 82, 82a, 82b, 83, 84, 85, and 105 in a FR of the heavy chainvariable region; (b) position 31, 61, 62, 63, 64, 65, and 97 in a CDR ofthe heavy chain variable region; (c) position 16, 18, 37, 41, 42, 45,65, 69, 74, 76, 77, 79, and 107 in a FR of the light chain variableregion; and (d) position 24, 25, 26, 27, 52, 53, 54, 55, and 56 in a CDRof the light chain variable region. In some embodiments, the antigen isa soluble antigen. In some embodiments, the method further comprisescomparing the KD of an antibody produced according to the method for itscorresponding antigen in an acidic pH (e.g., pH 5.8) and a neutral pH(e.g., pH 7.4). In further embodiments, the method comprises selectingan antibody that has a KD (acidic pH range (e.g., pH 5.8))/KD (neutralpH range (e.g., pH 7.4)), for the antigen of 2 or higher. In someembodiments, the method further comprises comparing the antigen bindingactivity of an antibody produced according to the method under a highion concentration (e.g., a hydrogen ion or calcium ion concentration)and a low ion concentration condition. In further embodiments the methodfurther comprises selecting an antibody that has a higher antigenbinding activity under a high ion concentration (e.g., 2-fold) thanunder a low ion concentration. In some embodiments, where the ionconcentration is calcium ion concentration, the high calcium ionconcentration may be selected between 100 μM and 10 mM, between 200 μMand 5 mM, between 400 μM and 3 mM, between 200 μM and 2 mM, or between400 μM and 1 mM. A concentration selected between 500 μM and 2.5 mM,which is close to the plasma (blood) concentration of calcium ion invivo, may be also preferred. In some embodiments, the low calcium ionconcentration may be selected between 0.1 μM and 30 between 0.2 μM and20 between 0.5 μM and 10 or between 1 μM and 5 or between 2 μM and 4 μM.A concentration selected between 1 μM and 5 which is close to theconcentration of calcium ion in early endosomes in vivo, may be alsopreferred. In some embodiments, the lower limit of the KD (low calciumion concentration condition)/KD (high calcium ion concentrationcondition) (e.g., KD (3 μM Ca)/KD (2 mM Ca)) value is 2 or more, 10 ormore, or 40 or more, and the upper limit thereof is 400 or less, 1000 orless, or 10000 or less. In alternative some embodiments, the lower limitof the kd (low calcium ion concentration condition)/kd (high calcium ionconcentration condition) (e.g., kd (3 μM Ca)/kd (2 mM Ca)) value is 2 ormore, 5 or more, 10 or more, or 30 or more, and the upper limit thereofis 50 or less, 100 or less, or 200 or less. In some embodiments, wherethe ion concentration is hydrogen ion concentration, low hydrogen ionconcentration (neutral pH range) may be selected from pH 6.7 to pH 10.0,from pH 6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0.The low hydrogen ion concentration may be preferably pH 7.4 which isclose to the in vivo pH in plasma (blood), but for the convenience ofmeasurement, for example, pH 7.0 may be used. In some embodiments, highhydrogen ion concentration (acidic pH range) may be selected from pH 4.0to pH 6.5, from pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5.The acidic pH range may be preferably pH 5.8 which is close to the invivo hydrogen ion concentration in the early endosome, but for theconvenience of measurement, for example, pH 6.0 may be used. In someembodiments, the lower limit of KD (acidic pH range)/KD (neutral pHrange) (e.g., KD (pH 5.8)/KD (pH 7.4)) is 2 or more, 10 or more, or 40or more, and the upper limit thereof is 400 or less, 1000 or less, or10000 or less. In some embodiments, the method further comprisescomparing the elimination of antigen from plasma after theadministration of an antibody produced according to the method ascompared to that when a reference antibody which differs only in that itdoes not include the modification(s) introduced according to the method,is administered. In further embodiments, the method further comprisesselecting an antibody produced according to the method that promoteselimination of the antigen from plasma (e.g., 2-fold) as compared to anantibody that does not contain the modifications introduced according tothe method. In some embodiments, the method further comprises comparingextracellular matrix-binding of the antibody produced according to themethod as compared to the antibody which differs only in that it doesnot include the modification(s) introduced according to the method. Infurther embodiments the method further comprises selecting an antibodyproduced according to the method that has increased extracellularmatrix-binding (e.g., 2-fold when bound to an antigen) as compared to anantibody which differs only in that it does not include themodification(s) introduced according to the method. In furtherembodiments, the antibodies produced according to the method(substantially) retain the antigen-binding activity when compared to theantibodies before modification or alteration of at least one amino acidresidue to increase pI (native antibodies (for example, native Igantibodies, preferably native IgG antibodies) or reference antibodies(e.g., antibodies before antibody modification, or prior to or duringlibrary construction)). In this case, “to (substantially) retain theantigen-binding activity” can mean to have an activity of at least 50%or more, preferably 60% or more, more preferably 70% or 75% or more, andstill more preferably 80%, 85%, 90%, or 95% or more as compared to thebinding activity of the antibodies before modification or alteration.

In an additional embodiment, Disclosure A relates to a method forproducing an antibody comprising an antigen-binding domain whoseantigen-binding activity changes according to ion concentrationconditions, wherein the method comprises modifying at least one aminoacid residue that may be exposed on the surface of a constant region ofan antibody so as to increase the isoelectric point (pI). In someembodiments, the amino acid residue modification comprises amodification selected from the group consisting of: (a) substitution ofa negatively charged amino acid residue with an uncharged amino acidresidue; (b) substitution of a negatively charged amino acid residuewith a positively charged amino acid residue; and (c) substitution of anuncharged amino acid residue with a positively charged amino acidresidue. In some embodiments, at least one modified amino acid residueis substituted with histidine. In further embodiments, the antibodycomprises a variable region and/or a constant region, and an amino acidresidue is modified in the variable region and/or the constant region.In further embodiments, at least one amino acid residue modifiedaccording to the method is in a position in a constant region selectedfrom the group consisting of position 196, 253, 254, 256, 258, 278, 280,281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356,358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401,402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according toEU numbering. In further embodiments, at least one amino acid residuemodified according to the method is in a position in a constant regionselected from the group consisting of position 254, 258, 281, 282, 285,309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 384,385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434,and 443, according to EU numbering. In yet further embodiments, at leastone amino acid residue modified according to the method is in a positionin a constant region selected from the group consisting of position 282,309, 311, 315, 342, 343, 384, 399, 401, 402, and 413, according to EUnumbering. In some embodiments, the method further comprises comparingthe antigen binding activity of an antibody produced according to themethod under a high ion concentration (e.g., a hydrogen ion or calciumion concentration) and a low ion concentration condition. In furtherembodiments the method further comprises selecting an antibody that hasa higher antigen binding activity under a high ion concentration thanunder a low ion concentration. In some embodiments, the method comprisescomparing the elimination of antigen from plasma after theadministration of an antibody produced according to the method ascompared to that when a reference antibody which differs only in that itdoes not include the modification(s) introduced according to the method,is administered. In further embodiments the method further comprisesselecting an antibody produced according to the method that promoteselimination of the antigen from plasma (e.g., 2-fold) as compared to anantibody that does not contain the modifications introduced according tothe method. In some embodiments, the method comprises comparingextracellular matrix-binding of the antibody produced according to themethod as compared to the antibody which differs only in that it doesnot include the modification(s) introduced according to the method. Infurther embodiments the method further comprises selecting an antibodyproduced according to the method that has increased extracellularmatrix-binding binding (e.g., 2-fold when bound to antigen) as comparedto an antibody which differs only in that it does not include themodification(s) introduced according to the method. In some embodiments,the method comprises comparing the Fc gamma receptor (FcγR)-bindingactivity under neutral pH (e.g., pH 7.4) of an antibody producedaccording to the method with that of a reference antibody comprising aconstant region of a native IgG. In further embodiments the methodcomprises selecting an antibody produced according to the method thathas enhanced FcγR-binding activity under a neutral pH (e.g., pH 7.4) ascompared to that of the reference antibody comprising a constant regionof a native IgG. In some embodiments, the selected antibody producedaccording to the method has enhanced FcγRIIb binding activity underneutral pH. In some embodiments, the selected antibody producedaccording to the method has binding activity towards one or moreactivating FcγR, preferably selected from the group consisting ofFcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa, and towardsFcγRIIb, and optionally the FcγRIIb-binding activity is maintained orenhanced and the binding activity to the activating FcγRs is decreased,as compared to those of a reference antibody which differs only in thatits constant region is that of a native IgG. In some embodiments, themethod further comprises comparing FcRn-binding activity under a neutralpH condition of the antibody produced according to the method ascompared to a reference antibody which differs only in that its constantregion is that of a native IgG. In further embodiments, the methodfurther comprises selecting an antibody produced according to the methodthat has increased FcRn-binding activity under a neutral pH condition ascompared to that of a reference antibody which differs only in that itsconstant region is that of a native IgG (e.g., 2-fold). In someembodiments, the antibodies produced according to the method(substantially) retain the antigen-binding activity when compared to theantibodies before modification or alteration of at least one amino acidresidue to increase pI (native antibodies (for example, native Igantibodies, preferably native IgG antibodies) or reference antibodies(e.g., antibodies before antibody modification, or prior to or duringlibrary construction)). In this case, “to (substantially) retain theantigen-binding activity” can mean to have an activity of at least 50%or more, preferably 60% or more, more preferably 70% or 75% or more, andstill more preferably 80%, 85%, 90%, or 95% or more as compared to thebinding activity of the antibodies before modification or alteration.

In additional embodiments, the method comprises modifying at least oneamino acid residue that may be exposed on the surface of a variableregion and constant region of an antibody so as to increase theisoelectric point (pI). In further embodiments, at least one amino acidresidue modified according to the method is in a position in a constantregion disclosed above. In further embodiments at least one amino acidresidue modified according to the method is in a position in a variableregion disclosed above. In further embodiments, at least one amino acidresidue modified according to the method is in a position in a constantregion disclosed above and at least one amino acid residue modifiedaccording to the method is in a position in a variable region disclosedabove. In some embodiments, the antigen is a soluble antigen. In someembodiments, the method further comprises comparing the KD of anantibody produced according to the method for its corresponding antigenin an acidic pH (e.g., pH 5.8) and a neutral pH (e.g., pH 7.4). Infurther embodiments, the method comprises selecting an antibody that hasa KD (acidic pH range)/KD (neutral pH range), for the antigen of 2 orhigher. In some embodiments, the method further comprises comparing theantigen binding activity of an antibody produced according to the methodunder a high ion concentration (e.g., a hydrogen ion or calcium ionconcentration) condition and a low ion concentration condition. Infurther embodiments, the method further comprises selecting an antibodythat has a higher antigen binding activity under a high ionconcentration than under a low ion concentration. In some embodiments,where the ion concentration is calcium ion concentration, the highcalcium ion concentration may be selected between 100 μM and 10 mM,between 200 μM and 5 mM, between 400 μM and 3 mM, between 200 μM and 2mM, or between 400 μM and 1 mM. A concentration selected between 500 μMand 2.5 mM may be also preferred. In some embodiments, the low calciumion concentration may be selected between 0.1 μM and 30 between 0.2 μMand 20 between 0.5 and 10 or between 1 μM and 5 or between 2 μM and 4μM. A concentration selected between 1 μM and 5 μM may be alsopreferred. In some embodiments, the lower limit of the KD (low calciumion concentration condition)/KD (high calcium ion concentrationcondition) (e.g., KD (3 μM Ca)/KD (2 mM Ca)) value is 2 or more, 10 ormore, or 40 or more, and the upper limit thereof is 400 or less, 1000 orless, or 10000 or less. In alternative some embodiments, the lower limitof the kd (low calcium ion concentration condition)/kd (high calcium ionconcentration condition) (e.g., kd (3 μM Ca)/kd (2 mM Ca)) value is 2 ormore, 5 or more, 10 or more, or 30 or more, and the upper limit thereofis 50 or less, 100 or less, or 200 or less. In some embodiments, wherethe ion concentration is hydrogen ion concentration, low hydrogen ionconcentration (neutral pH range) may be selected from pH 6.7 to pH 10.0,from pH 6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0.The low hydrogen ion concentration may be preferably pH 7.4 which isclose to the in vivo pH in plasma (blood), but for the convenience ofmeasurement, for example, pH 7.0 may be used. In some embodiments, highhydrogen ion concentration (acidic pH range) may be selected from pH 4.0to pH 6.5, from pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5.The acidic pH range may be pH 5.8 or pH 6.0, for example. In someembodiments, the lower limit of KD (acidic pH range)/KD (neutral pHrange) (e.g., KD (pH 5.8)/KD (pH 7.4)) is 2 or more, 10 or more, or 40or more, and the upper limit thereof is 400 or less, 1000 or less, or10000 or less.

In some embodiments, the method further comprises comparing theelimination of antigen from plasma after the administration of anantibody produced according to the method as compared to that when areference antibody which differs only in that it does not include themodification(s) introduced according to the method is administered. Infurther embodiments, the method further comprises selecting an antibodyproduced according to the method that promotes elimination of theantigen from plasma (e.g., 2-fold) as compared to an antibody that doesnot contain the modifications introduced according to the method. Insome embodiments, the method further comprises comparing extracellularmatrix-binding of the antibody produced according to the method ascompared to the antibody which differs only in that it does not includethe modification(s) introduced according to the method. In furtherembodiments, the method further comprises selecting an antibody producedaccording to the method that has increased extracellular matrix-bindingbinding (e.g., 5-fold when complexed with antigen) as compared to anantibody which differs only in that it does not include themodification(s) introduced according to the method. In some embodiments,the method further comprises comparing the Fc gamma receptor(FcγR)-binding activity under neutral pH (e.g., pH 7.4) of an antibodyproduced according to the method with that of a reference antibodycomprising a constant region of a native IgG. In further embodiments,the method comprises selecting an antibody produced according to themethod that has enhanced FcγR-binding activity under a neutral pH (e.g.,pH 7.4) as compared to that of the reference antibody comprising aconstant region of a native IgG. In some embodiments, the selectedantibody produced according to the method has enhanced FcγRIIb bindingactivity under neutral pH. In some embodiments, the selected antibodyproduced according to the method has binding activity towards one ormore activating FcγR, preferably selected from the group consisting ofFcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa, and towardsFcγRIIb, and optionally the FcγRIIb-binding activity is maintained orenhanced and the binding activity to the activating FcγRs is decreased,as compared to those of a reference antibody which differs only in thatits constant region is that of a native IgG. In some embodiments, themethod further comprises comparing FcRn-binding activity under a neutralpH condition of the antibody produced according to the method ascompared to a reference antibody which differs only in that its constantregion is that of a native IgG. In further embodiments, the methodfurther comprises selecting an antibody produced according to the methodthat has increased FcRn-binding activity under a neutral pH condition(e.g., 2-fold) as compared to that of a reference antibody which differsonly in that its constant region is that of a native IgG. In furtherembodiments, the antibodies produced according to the method(substantially) retain the antigen-binding activity when compared to theantibodies before modification or alteration of at least one amino acidresidue to increase pI (native antibodies (for example, native Igantibodies, preferably native IgG antibodies) or reference antibodies(e.g., antibodies before antibody modification, or prior to or duringlibrary construction)). In this case, “to (substantially) retain theantigen-binding activity” can mean to have an activity of at least 50%or more, preferably 60% or more, more preferably 70% or 75% or more, andstill more preferably 80%, 85%, 90%, or 95% or more as compared to thebinding activity of the antibodies before modification or alteration.

In an alternative embodiment, Disclosure A relates to an antibodyobtained by the above-described method of Disclosure A for producing orscreening antibodies.

In an alternative embodiment, Disclosure A relates to a composition orpharmaceutical composition comprising an antibody of Disclosure Adescribed above. In one embodiment, the pharmaceutical composition ofDisclosure A may be a pharmaceutical composition for acceleratingantigen elimination from a biological fluid (preferably, plasma, etc.)of subjects and/or for increasing the extracellular matrix binding (whenan antibody of Disclosure A is administered to (applied to) the subject(preferably, in vivo)). The pharmaceutical composition of Disclosure Amay optionally contain a pharmaceutically acceptable carrier. Herein,pharmaceutical compositions may typically refer to agents for use intreatment, prevention, diagnosis, or examination of diseases.

The compositions or pharmaceutical compositions of Disclosure A can besuitably formulated. In some embodiments, they can be used parenterally,for example, in a form of a sterile solution or suspension for injectionin water or any other pharmaceutically acceptable liquid. Thecompositions can be suitably formulated at a unit dose required forgenerally accepted pharmaceutical practice, by appropriately combiningwith pharmaceutically acceptable carriers or media. Suchpharmaceutically acceptable carriers or media include, but are notlimited to, sterile water, physiological saline, vegetable oils,emulsifiers, suspending agents, surfactants, stabilizers, flavoringagents, excipients, vehicles, preservatives, and binding agents. Theamount of active ingredient in the compositions may be adjusted in sucha way that the dose falls within an appropriate pre-determined range.

In some embodiments, the compositions or pharmaceutical compositions ofDisclosure A can be administered parenterally. The compositions orpharmaceutical compositions may be appropriately prepared as, forexample, an injectable, transnasal, transpulmonary, or transdermalcomposition. The compositions or pharmaceutical compositions may beadministered systemically or locally, for example, by intravenousinjection, intramuscular injection, intraperitoneal injection, orsubcutaneous injection.

In some embodiments, the disclosure provides antibodies whose pI isincreased by modifying at least one amino acid residue that can beexposed on the surface (antibodies with increased pI); methods forproducing these antibodies; or use of these antibodies to enhanceantigen elimination from plasma (when the antibodies are administered tothe subjects in vivo). It can be understood that the scope of DisclosureA described herein and the contents described in the counterpartExamples herein can be appropriately applied to such embodiments. Inother embodiments, the disclosure provides antibodies whose pI isdecreased by modifying at least one amino acid residue that can beexposed on the surface (“antibodies with decreased pI”); methods forproducing these antibodies; or use of these antibodies to improve plasmaretention (when the antibodies are administered to the subjects invivo). The inventors have revealed that cellular internalization of anantibody can be enhanced by increasing its pI by introducing specificamino acid mutations into specific sites in the amino acid sequence ofthe constant region. Those of ordinary skill in the art can understandthat antibody plasma retention can be prolonged as the pI has beenreduced to suppress cellular internalization of the antibody byintroducing amino acids with a different side-chain charge property intothe sites described above. It can be understood that the scope ofDisclosure A described herein and the contents described in thecounterpart Examples herein can be appropriately applied to suchembodiments.

In one embodiment, the disclosure provides a method for producing amodified antibody, whose half life in plasma is prolonged or reduced, ascompared to that before the modification of the antibody, wherein themethod comprises: (a) modifying a nucleic acid encoding the antibodybefore the modification to change the charge of at least one amino acidresidue located at a position selected from the group consisting ofposition 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286,306, 307, 308, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359,361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402,413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EUnumbering; (b) culturing a host cell to express the modified nucleicacid and to produce the antibody; and (c) collecting the producedantibody from the host cell culture.

An additional embodiment provides a method for prolonging or reducingthe half-life of an antibody in plasma wherein the method comprisesmodifying at least one amino acid residue located at a position selectedfrom the group consisting of position 196, 253, 254, 256, 257, 258, 278,280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315, 327, 330, 342,343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388,389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434,and 443, according to EU numbering.

These methods may further comprise determining that the half life inplasma of the collected and/or modified antibody is prolonged orreduced, as compared to that before the modification of the antibody.

The change of charge may be achieved by amino acid substitution(s). Insome embodiments, the substituted amino acid residue(s) may be selectedfrom the group consisting of the amino acid residues of group (a) and(b) below, but is not limited thereto: (a) Glu (E) and Asp (D); and (b)Lys (K), Arg (R) and His (H).

In some embodiments, the antibody may be an Ig-type antibody such as anIgG antibody. In some embodiments, the antibody may be a chimericantibody, humanized antibody, or human antibody. In some embodiments,the antibody may be a multispecific antibody such as a bispecificantibody.

Disclosure B

In non-limited embodiments, Disclosure B relates to Fc region variants,uses thereof, and production methods thereof.

Within the scope of Disclosures A and B described herein, an “Fc regionvariant” may refer, for example, to an Fc region modified from the Fcregion of a native IgG antibody by modifying at least one amino acidwith another amino acid, or may refer to an Fc region modified from suchan Fc region variant by additionally modifying at least one amino acidwith another amino acid. Herein, such Fc region variants include notonly Fc regions that have been introduced with the amino acidmodification but also Fc regions containing the same amino acid sequenceas an aforementioned Fc region.

In an alternative embodiment, Disclosure B relates to Fc region variantscontaining an FcRn-binding domain which contains Ala at position 434;any one of Glu, Arg, Ser, and Lys at position 438; and any one of Glu,Asp, and Gln at position 440, according to EU numbering (within thescope of Disclosure B described herein, such an Fc region variant isalso referred to as a “novel Fc region variant” for descriptivepurposes).

In practice, Fc region variants of Disclosure B can be incorporated intovirtually any antibody (e.g., multispecific antibodies such asbispecific antibodies) regardless of the type of the target antigen. Forexample, Anti-factor IXa/factor X bispecific antibodies can be producedusing such Fc region variants as shown in Example 20 (e.g., F8M-F1847mv[F8M-F1847mv1 (SEQ ID NO:323) and F8M-F1847mv2 (SEQ ID NO:324) as theheavy chains and F8ML (SEQ ID NO:325) as the light chain]; F8M-F1868mv[F8M-F1868mv1 (SEQ ID NO:326) and F8M-F1868mv2 (SEQ ID NO:327) as theheavy chains and F8ML (SEQ ID NO:325) as the light chain]; andF8M-F1927mv [F8M-F1927mv1 (SEQ ID NO:328) and F8M-F1927mv2 (SEQ IDNO:329) as the heavy chains and F8ML (SEQ ID NO:325) as the lightchain]).

As described above, WO2013/046704 reports that Fc region variants thathave been introduced with a mutation to increase their FcRn bindingunder acidic conditions in combination with a specific mutation (arepresentative example is dual-residue mutation Q438R/S440E according toEU numbering) exhibit significantly reduced binding to rheumatoidfactor. However, WO2013/046704 does not describe that the Fc regionvariants whose rheumatoid factor binding has been reduced due to theQ438R/S440E modification are superior in plasma retention as compared toantibodies with a native Fc region. Thus, there is a demand for safe andmore advantageous Fc region variants that allow improved plasmaretention, but do not bind to pre-existing ADA. The inventors discloseherein safe and more advantageous Fc region variants that allow improvedplasma retention, but do not bind to anti-drug antibodies (pre-existingADA, etc.). In particular, it is first disclosed herein thatsurprisingly, Fc region variants that contain combined mutations ofamino acid residues, which are a substitution of Ala (A) for the aminoacid at position 434 according to EU numbering and a specificdual-residue mutation (a representative example is Q438R/S440E), arepreferable for prolonging antibody retention in plasma while maintaininga significantly reduced binding to rheumatoid factor.

Thus, the novel Fc region variants of Disclosure B disclosed hereinprovides an advantageous and surprising improvement over the Fc regionvariants described in WO2013/046704, which is incorporated herein byreference in their entirety.

In one embodiment, Disclosure B provides novel combinations of aminoacid substitutions in the FcRn-binding domain, which increase theFcRn-binding activity of antibodies in an acidic pH range and in aneutral pH range, in particular, in an acidic pH range.

In one embodiment, an Fc region variant of Disclosure B contains Ala atposition 434; any one of Glu, Arg, Ser, and Lys at position 438; and anyone of Glu, Asp, and Gln at position 440, according to EU numbering; andmore preferably contain Ala at position 434; either Arg or Lys atposition 438; and either Glu or Asp at position 440, according to EUnumbering. Preferably, the Fc region variant of Disclosure Badditionally contains either Ile or Leu at position 428, and/or any oneof Ile, Leu, Val, Thr, and Phe at position 436, according to EUnumbering. More preferably the Fc region variant contains Leu atposition 428, and/or either Val or Thr at position 436, according to EUnumbering.

In one embodiment, the Fc region variant of Disclosure B can be an Fcregion variant of a native Ig antibody, and more preferably the Fcregion variant of a native IgG (IgG1, IgG2, IgG3, or IgG4 type)antibody. The native Fc region is partly described within the scope ofDisclosures A and B, herein. More specifically, in Disclosure B, thenative Fc region can refer to an unmodified or naturally-occurring Fcregion, and preferably, an unmodified or naturally-occurring Fc regionof a native Ig antibody whose Fc region amino acid residues remainunmodified. The antibody origin of the Fc region can be an Ig such asIgM or IgG, for example, human IgG1, IgG2, IgG3, or IgG4. In oneembodiment, it may be human IgG1. Meanwhile, a (reference) antibodycomprising a native Fc region can refer to an antibody comprising anunmodified or naturally-occurring Fc region.

Positions 428, 434, 438, and 440 are common to Fc regions of all nativehuman IgG1, IgG2, IgG3, and IgG4 antibodies. However, at position 436 inthe Fc region, native human IgG1, IgG2, and IgG4 antibodies share Tyr(Y) whereas native human IgG3 antibody has Phe (F). On the other hand,Stapleton et al. (Nature Comm. 599 (2011) reported that human IgG3allotypes containing the amino acid substitution of R435H according toEU numbering have a plasma half-life in human comparable to that ofIgG1. Thus, the inventors also conceived that plasma retention could beimproved by increasing FcRn binding under an acidic condition byintroducing the R435H amino acid substitution in combination with theamino acid substitution at position 436.

WO2013/046704 also specifically reported dual amino acid residuesubstitutions of Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440Daccording to EU numbering, which result in a significant reduction ofthe rheumatoid factor binding when combined with an amino acidsubstitution that can increase the FcRn binding under an acidiccondition.

Thus, in a preferred embodiment, the FcRn-binding domain of an Fc regionvariant of Disclosure B may contain a combination of substituted aminoacid positions selected from the group consisting of: (a)N434A/Q438R/S440E; (b) N434A/Q438R/S440D; (c) N434A/Q438K/S440E; (d)N434A/Q438K/S440D; (e) N434A/Y436T/Q438R/S440E; (f)N434A/Y436T/Q438R/S440D; (g) N434A/Y436T/Q438K/S440E; (h)N434A/Y436T/Q438K/S440D; (i) N434A/Y436V/Q438R/S440E; (j)N434A/Y436V/Q438R/S440D; (k) N434A/Y436V/Q438K/S440E; (1)N434A/Y436V/Q438K/S440D; (m) N434A/R435H/F436T/Q438R/S440E; (n)N434A/R435H/F436T/Q438R/S440D; (o) N434A/R435H/F436T/Q438K/S440E; (p)N434A/R435H/F436T/Q438K/S440D; (q) N434A/R435H/F436V/Q438R/S440E; (r)N434A/R435H/F436V/Q438R/S440D; (s) N434A/R435H/F436V/Q438K/S440E; (t)N434A/R435H/F436V/Q438K/S440D; (u) M428L/N434A/Q438R/S440E; (v)M428L/N434A/Q438R/S440D; (w) M428L/N434A/Q438K/S440E; (x)M428L/N434A/Q438K/S440D; (y) M428L/N434A/Y436T/Q438R/S440E; (z)M428L/N434A/Y436T/Q438R/S440D; (aa) M428L/N434A/Y436T/Q438K/S440E; (ab)M428L/N434A/Y436T/Q438K/S440D; (ac) M428L/N434A/Y436V/Q438R/S440E; (ad)M428L/N434A/Y436V/Q438R/S440D; (ae) M428L/N434A/Y436V/Q438K/S440E; (af)M428L/N434A/Y436V/Q438K/S440D; (ag)L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and (ah)L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E, accordingto EU numbering.

In a further preferred embodiment, the FcRn-binding domain of an Fcregion variant of Disclosure B may contain a combination of substitutedamino acids selected from the group consisting of: (a)N434A/Q438R/S440E; (b) N434A/Y436T/Q438R/S440E; (c)N434A/Y436V/Q438R/S440E; (d) M428L/N434A/Q438R/S440E; (e)M428L/N434A/Y436T/Q438R/S440E; (f) M428L/N434A/Y436V/Q438R/S440E; (g)L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and (h)L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E, accordingto EU numbering.

In one embodiment, it is preferable that the FcRn-binding activity of anFc region variant of Disclosure B has been increased under an acidic pHcondition as compared to the Fc region of a native IgG.

An increase in the FcRn-binding activity (binding affinity) of anFcRn-binding domain in a pH range may correspond to an increase of themeasured FcRn-binding activity (binding affinity) when compared to themeasured FcRn-binding activity (binding affinity) of a nativeFcRn-binding domain. In this case, KD (native Fc region)/KD (an Fcregion variant of Disclosure B), which represents a difference in thebinding activity (binding affinity), may be at least 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 70-fold,80-fold, 100-fold, 500-fold, or 1000-fold. Such an increase may occur inan acidic pH range and/or in a neutral pH range; however, the increasein an acidic pH range can be preferred from the viewpoint of the actionmechanism for Disclosure B.

In some embodiments, the FcRn-binding activity (for example, at pH 6.0and 25° C.) of an Fc region variant of Disclosure B whose FcRn-bindingactivity has been increased in an acidic pH range is greater than thatof the Fc region of a native IgG, for example, by 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 200-fold, 500-fold,1000-fold or more. In some embodiments, the increased FcRn-bindingactivity of an Fc region variant in an acidic pH range may be greaterthan the FcRn-binding activity of the Fc region of a native IgG by atleast 5-fold or at least 10-fold.

Manipulation of the FcRn-binding domain by introducing amino acidsubstitutions can occasionally reduce antibody stability(WO2007/092772). Proteins with poor stability tend to aggregate easilyduring storage, and the stability of pharmaceutical proteins is highlyimportant in production of pharmaceutical agents. Thus, the decrease instability caused by substitutions in the Fc region can lead todifficulty in developing stable antibody preparations (WO2007/092772).

The purity of pharmaceutical proteins in terms of monomer andhigh-molecular-weight species is also important in developingpharmaceutical agents. After purification with Protein A, wild-type IgG1does not contain a significant amount of high-molecular-weight species,whereas manipulation of the FcRn-binding domain by introducingsubstitutions can produce a large amount of high-molecular-weightspecies. In this case, such high-molecular-weight species may have to beremoved from the drug substance by purification steps.

Amino acid substitutions in antibodies can result in negativeconsequences, such as an increase in the immunogenicity of therapeuticantibodies which in turn can cause a cytokine storm and/or production ofanti-drug antibodies (ADAs). The clinical utility and efficacy oftherapeutic antibodies can be limited by ADAs, since they affect theefficacy and pharmacokinetics of therapeutic antibodies and sometimeslead to serious side effects. Many factors influence the immunogenicityof therapeutic antibodies, and the presence of effector T-cell epitopesis one of the factors. Likewise, the presence of pre-existing antibodiesagainst a therapeutic antibody can also be problematic. An example ofsuch pre-existing antibody is rheumatoid factor (RF), an auto-antibody(an antibody directed against a self-protein) against the Fc portion ofan antibody (i.e., IgG). Rheumatoid factor is found in particular inpatients with systemic lupus erythematosus (SLE) or rheumatoidarthritis. In arthritis patients, RF and IgG join to form immunecomplexes that contribute to the disease process. Recently, a humanizedanti-CD4 IgG1 antibody with a Asn434His mutation has been reported toelicit significant rheumatoid factor binding (Zheng et al., Clin.Pharmacol. Ther. 89(2):283-290 (2011)). Detailed studies have confirmedthat the Asn434His mutation in human IgG1 increases the binding ofrheumatoid factor to the Fc region of the antibody as compared to theparental human IgG1.

RF is a polyclonal auto-antibody against human IgG. The RF epitope inthe human IgG sequence varies among clones; however, the RF epitopeseems to be located in the CH2/CH3 interface region as well as in theCH3 domain which may overlap with the FcRn-binding epitope. Thus,mutations to increase the FcRn-binding activity at a neutral pH maypossibly increase the binding activity to specific RF clones as well.

In the context of Disclosure B, the term “anti-drug antibody” or “ADA”can refer to an endogenous antibody that has binding activity to anepitope located on a therapeutic antibody and thus can bind to thetherapeutic antibody. The term “pre-existing anti-drug antibody” or“pre-existing ADA” can refer to an anti-drug antibody that is presentand detectable in the blood of a patient prior to administration of thetherapeutic antibody to the patient. In some embodiments, thepre-existing ADA is a human antibody. In further embodiments, thepre-existing ADA is rheumatoid factor.

The binding activity of an antibody Fc region (variant) against apre-existing ADA can be, for example, represented byelectrochemiluminescence (ECL) response at an acidic pH and/or at aneutral pH. The ECL assay is described, for example, in Moxness et al.(Clin Chem. 51:1983-1985 (2005)) and in Example 6. Assays can beperformed, for example, under the conditions of MES buffer and 37° C.The antigen-binding activity of antibodies can be determined by, forexample, BIACORE® analysis.

The binding activity to a pre-existing ADA can be assessed at anytemperature from 10° C. to 50° C. In some embodiments, the bindingactivity (binding affinity) of a human Fc region to a human pre-existingADA is determined at a temperature of 15° C. to 40° C., for example,such as between 20° C. to 25° C., or 25° C. In a further embodiment, theinteraction between a human pre-existing ADA and a human Fc region ismeasured at pH 7.4 (or pH 7.0) and 25° C.

Within the scope of Disclosure B described herein, the binding activityto (pre-existing) ADA has been significantly increased or an equivalentexpression may mean that the measured (pre-existing) ADA-bindingactivity (binding affinity) (i.e., KD) of an Fc region variant ofDisclosure B or an antibody containing it has been increased, forexample, by 0.55-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold,1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, or 2.3-fold or more, ascompared to the measured (pre-existing) ADA-binding activity (bindingaffinity) of a reference Fc region variant or a reference antibodycontaining the reference Fc region variant. Such an increase in thebinding activity to a pre-existing ADA can be observed in an individualpatient or in a patient group.

In one embodiment, as used in the context of Disclosure B, the term“patients” or “a patient” is not limited, and can include all humanswith a disease who are under treatment with a therapeutic antibody. Thepatients may be humans affected with auto-immune disease, such as anarthritic disease or systemic erythematosus (SLE). The arthritic diseasecan include rheumatoid arthritis.

In one embodiment of Disclosure B, the binding activity to apre-existing ADA is significantly increased in an individual patient maymean that the binding activity of an antibody comprising an Fc regionvariant (e.g., therapeutic antibody) to a pre-existing ADA measured in apatient has been increased by at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, or at least 60% or more when compared to thebinding activity of a reference antibody to the pre-existing ADA.Alternatively, this may mean that the ECL reaction for the antibody ispreferably above 250, or at least 500, or at least 1000, or even atleast 2000. Preferably, this increase may be an increase relative to areference antibody whose ECL reaction is less than 500 or 250.Specifically, between the binding activity of a reference antibody to apre-existing ADA and such binding activity of an antibody having an Fcregion variant, the ECL reaction preferably ranges from less than 250 toat least 250, less than 250 to at least 500, less than 500 to 500 ormore, less than 500 to 1000 or more, or less than 500 to at least 2000,without being limited thereto.

In one embodiment, the binding activity to a pre-existing ADA isincreased can mean that in a group of patients, the measured proportionof patients who have an ECL reaction of at least 500 (preferably, atleast 250) for an antibody comprising an Fc region variant with (a)increased binding activity to FcRn at an acidic pH and (b) increasedbinding activity to a pre-existing ADA at a neutral pH is elevated ascompared to the proportion of patients who have an ECL reaction of atleast 500 (preferably, at least 250 or more) for a reference antibody,for example, by at least 10%, at least 20%, at least 30%, at least 40%,at least 50% when compared to the proportion of patients who have an ECLreaction for a reference antibody.

In one embodiment of Disclosure B, the binding activity to apre-existing ADA decreases can mean that the measured binding activity(i.e., KD or ECL reaction) of an antibody comprising an Fc regionvariant decreases as compared to that of a reference antibody. Suchdecrease can be observed in an individual patient or in a group ofpatients. The affinity of an antibody comprising an Fc region variantfor a pre-existing ADA at a neutral pH significantly decreases in eachpatient can mean that the measured binding activity to a pre-existingADA at a neutral pH measured in the patient is decreased as compared tothe binding activity of a reference antibody to the pre-existing ADAmeasured at the neutral pH, for example, by at least 10%, at least 20%,at least 30%, at least 40%, or at least 50%.

Alternatively, the binding activity of an antibody containing an Fcregion variant to a pre-existing ADA significantly decreases in anindividual patient can mean that the ECL reaction for the antibody thatused to be 500 or more (preferably, 1000 or more, or 2000 or more) ismeasured to be less than 500, preferably less than 250 as compared tothe ECL reaction for a reference antibody, for example.

In a preferred embodiment, the Fc region variants of Disclosure B andantibodies comprising them have low binding activity to a pre-existingADA at a neutral pH. Specifically, it is preferable that the bindingactivity of antibodies containing the Fc region variants of Disclosure Bto a pre-existing ADA at a neutral pH is lower than or has notsignificantly been increased, as compared to the binding activity of areference antibody containing the Fc region of a native IgG to thepre-existing ADA at a neutral pH (e.g., pH 7.4). The binding activity(binding affinity) to a pre-existing ADA is low or the affinity is atthe baseline level can mean an ECL reaction of less than 500, or lessthan 250 in an individual patient, but is not limited thereto. Thebinding activity to a pre-existing ADA is low in a group of patients canmean that the ECL reaction is less than 500 in 90%, preferably 95%, andmore preferably 98% of the patients in the group, for example.

It can be preferable to select Fc region variants of Disclosure B orantibodies containing them, whose binding activity to a (pre-existing)ADA in plasma at a neutral pH is not significantly increased, and whoseFcRn-binding activity at a neutral pH and/or at an acidic pH isincreased. Preferably the FcRn-binding activity at an acidic pH (e.g.,pH 5.8) is increased. In one embodiment, the Fc region variantspreferably do not have a significantly increased binding activity to ADAunder a neutral pH condition (e.g., pH 7.4) as compared to the Fc regionof a native IgG, and the ADA may be a pre-existing ADA, preferablyrheumatoid factor (RF).

In one embodiment, it can be preferable that the Fc region variants ofDisclosure B have an increased FcRn-binding activity under an acidic pHcondition as compared to the Fc region of a native IgG, and as a resultthey exhibit reduced clearance (CL) in plasma, prolonged retention timein plasma, or prolonged half-life in plasma (t1/2). Their correlation isknown in the art.

In one embodiment, it can be preferable that the Fc region variants ofDisclosure B have an increased FcRn-binding activity under an acidic pHcondition but do not have a significantly increased ADA-binding activityunder a neutral pH condition as compared to the Fc region of a nativeIgG, and they exhibit reduced clearance (CL) in plasma, prolongedretention time in plasma, or prolonged half-life in plasma (t1/2). TheADA may be a pre-existing ADA, preferably rheumatoid factor (RF).

In one embodiment, the Fc region variants of Disclosure B areadvantageous, since their plasma retention is improved as compared to areference Fc region variant comprising a combination of amino acidsubstitutions N434Y/Y436V/Q438R/S440E according to EU numbering.

Examples 5 to 7 compare the plasma retention of two Fc region variants:Fc region variant F1718 (Fc region with mutations introduced at foursites: N434Y/Y436V/Q438R/S440E) described in WO2013/046704 and novel Fcregion variant F1848m (introduced with mutations at four sites:N434A/Y436V/Q438R/S440E). Difference in amino acid mutation between thetwo Fc region variants is only at position 434 according to EUnumbering, where the introduced amino acid mutation is Y (tyrosine) forF1718 and A (alanine) for F1848m. Nevertheless, when compared to anative IgG1, F1848m exhibited improved plasma retention while F1718showed no such improvement in plasma retention (see Example (7-2)).Thus, the Fc region variants of Disclosure B can preferably haveimproved plasma retention as compared to reference Fc region variantscontaining the combination of amino acid substitutionsN434Y/Y436V/Q438R/S440E. The experimental results described in Examples(5-2) and (7-3) herein demonstrate that among various Fc regionvariants, F1847m, F1886m, F1889m, and F1927m are further improved inplasma retention time than F1848m. Thus, those of ordinary skill in theart can appreciate that Fc region variants of Disclosure B comprisingF1847m, F1886m, F1889m, or F1927m, as well as F1848m have improvedplasma retention as compared to reference Fc region variants containingthe substitutions N434Y/Y436V/Q438R/S440E.

The binding to FcγR or a complement protein can also have an unfavorableimpact (for example, inappropriate platelet activation). Fc regionvariants that do not bind to effector receptors such as the FcγRIIareceptor can be safer and/or more advantageous. In some embodiments, theFc region variants of Disclosure B have only a weak effectorreceptor-binding activity or do not bind to effector receptors. Examplesof effector receptors include, activating FcγR, particularly FcγRI,FcγRII, and FcγRIII. FcγRI includes FcγRIa, FcγRIb, and FcγRIc, andsubtypes thereof. FcγRII includes FcγRIIa (having two allotypes: R131and H131) and FcγRIIb. FcγRIII includes FcγRIIIa (which has twoallotypes: V158 and F158) and FcγRIIIb (which has two allotypes:FcγIIIb-NA1 and FcγIIIb-NA2). Antibodies that have only a weak effectorreceptor-binding activity or do not bind to the receptors include, forexample, antibodies containing a silent Fc region and antibodies that donot have an Fc region (for example, Fab, F(ab)′₂, scFv, sc(Fv)₂, anddiabodies).

Examples of Fc regions which have only a weak or no effectorreceptor-binding activity are described, for example, in Strohl et al.(Curr. Op. Biotech. 20(6):685-691 (2009)), and specifically include, forexample, deglycosylated Fc regions (N297A and N297Q), and silent Fcregions resulting from manipulation of Fc regions to silence theireffector functions (or to suppress immunity) (IgG1-L234A/L235A,IgG1-H268Q/A330S/P331S, IgG1-C226S/C229S,IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S,IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S, IgG4-L235A/G237A/E318A,and IgG4-L236E). WO2008/092117 describes antibodies comprising a silentFc region that contains a substitution of G236R/L328R, L235G/G236R,N325A/L328R, or N325L/L328R, according to EU numbering. WO2000/042072describes antibodies comprising a silent Fc region that containssubstitutions at one or more of positions EU233 (position 233 accordingto EU numbering), EU234, EU235, and EU237. WO2009/011941 describesantibodies comprising a silent Fc region that lacks the residues ofEU231 to EU238. Davis et al. (J. Rheum. 34(11):2204-2210 (2007))describes antibodies with a silent Fc region containing substitutionsC220S/C226S/C229S/P238S. Shields et al. (J. Biol. Chem. 276(9):6591-6604(2001)) describes antibodies comprising a silent Fc region containingsubstitution D265A. Modification of these amino acid residues may alsobe appropriately introduced into the Fc region variants of Disclosure B.

The expression “weak binding to effector receptors” can mean that theeffector receptor-binding activity is, for example, 95% or less,preferably 90% or less, 85% or less, 80% or less, 75% or less, morepreferably 70% or less, 65% or less, 60% or less, 55% or less, 50% orless, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less,20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% orless, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1%or less than that of a native IgG or an antibody containing a native IgGFc region.

The “silent Fc region” is an Fc region variant containing one or moreamino acid substitutions, insertions, additions, deletions, and othersthat reduce binding to effector receptors as compared to a native Fcregion. Since the effector receptor-binding activity can be reducedconsiderably, such silent Fc regions may no longer bind to the effectorreceptors. The silent Fc regions may include, for example, Fc regionscontaining amino acid substitutions at one or more positions selectedfrom the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239,EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298,EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332. Modificationof these amino acid positions may also be appropriately introduced intothe Fc region variants of Disclosure B.

In further embodiments, the silent Fc region has a substitution at oneor more positions selected from the group consisting of: EU234, EU235,EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271,EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329,EU331, and EU332, and preferably the group consisting of: EU235, EU237,EU238, EU239, EU270, EU298, EU325, and EU329, wherein the substitutionis with an amino acid residue selected from the listing below:

The amino acid at position EU234 is preferably substituted with an aminoacid selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu,Gly, His, Lys, Met, Phe, Pro, Ser, and Thr.

The amino acid at position EU235 is preferably substituted with an aminoacid selected from the group consisting of Ala, Asn, Asp, Gln, Glu, Gly,His, Ile, Lys, Met, Pro, Ser, Thr, Val, and Arg.

The amino acid at position EU236 is preferably substituted with an aminoacid selected from the group consisting of Arg, Asn, Gln, His, Leu, Lys,Met, Phe, Pro, and Tyr.

The amino acid at position EU237 is preferably substituted with an aminoacid selected from the group consisting of Ala, Asn, Asp, Gln, Glu, His,Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, and Arg.

The amino acid at position EU238 is preferably substituted with an aminoacid selected from the group consisting of Ala, Asn, Gln, Glu, Gly, His,Ile, Lys, Thr, Trp, and Arg.

The amino acid at position EU239 is preferably substituted with an aminoacid selected from the group consisting of Gln, His, Lys, Phe, Pro, Trp,Tyr, and Arg.

The amino acid at position EU265 is preferably substituted with an aminoacid selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His,Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position EU266 is preferably substituted with an aminoacid selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu,Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, and Tyr.

The amino acid at position EU267 is preferably substituted with an aminoacid selected from the group consisting of Arg, His, Lys, Phe, Pro, Trp,and Tyr.

The amino acid at position EU269 is preferably substituted with an aminoacid selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position EU270 is preferably substituted with an aminoacid selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position EU271 is preferably substituted with an aminoacid selected from the group consisting of Arg, His, Phe, Ser, Thr, Trp,and Tyr.

The amino acid at position EU295 is preferably substituted with an aminoacid selected from the group consisting of Arg, Asn, Asp, Gly, His, Phe,Ser, Trp, and Tyr.

The amino acid at position EU296 is preferably substituted with an aminoacid selected from the group consisting of Arg, Gly, Lys, and Pro.

The amino acid at position EU297 is preferably substituted with Ala.

The amino acid at position EU298 is preferably substituted with an aminoacid selected from the group consisting of Arg, Gly, Lys, Pro, Trp, andTyr.

The amino acid at position EU300 is preferably substituted with an aminoacid selected from the group consisting of Arg, Lys, and Pro.

The amino acid at position EU324 is preferably substituted with eitherLys or Pro.

The amino acid at position EU325 is preferably substituted with an aminoacid selected from the group consisting of Ala, Arg, Gly, His, Ile, Lys,Phe, Pro, Thr, Trp, Tyr, and Val.

The amino acid at position EU327 is preferably substituted with an aminoacid selected from the group consisting of Arg, Gln, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position EU328 is preferably substituted with an aminoacid selected from the group consisting of Arg, Asn, Gly, His, Lys, andPro.

The amino acid at position EU329 is preferably substituted with an aminoacid selected from the group consisting of Asn, Asp, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, and Arg.

The amino acid at position EU330 is preferably substituted with eitherPro or Ser.

The amino acid at position EU331 is preferably substituted with an aminoacid selected from the group consisting of Arg, Gly, and Lys.

The amino acid at position EU332 is preferably substituted with an aminoacid selected from the group consisting of Arg, Lys, and Pro.

The silent Fc region preferably may contain a substitution with eitherLys or Arg at EU235, a substitution with either Lys or Arg at EU237, asubstitution with either Lys or Arg at EU238, a substitution with eitherLys or Arg at EU239, a substitution with Phe at EU270, a substitutionwith Gly at EU298, a substitution with Gly at EU325, or a substitutionwith either Lys or Arg at EU329. More preferably, the silent Fc regionmay contain a substitution with arginine at EU235 or a substitution withlysine at EU239. Even more preferably, the silent Fc region may containL235R/S239K substitutions. Modification of these amino acid residues mayalso be appropriately introduced into the Fc region variants ofDisclosure B.

In one embodiment, antibodies comprising an Fc region variant ofDisclosure B have only a weak complement protein-binding activity or donot bind to complement proteins. In some embodiments, the complementprotein is C1q. In some embodiments, the weak complement protein-bindingactivity refers to a complement protein-binding activity reduced by10-fold or more, 50-fold or more, or 100-fold or more when compared tothe complement protein-binding activity of a native IgG or an antibodycontaining a native IgG Fc region. The complement protein-bindingactivity of an Fc region can be reduced by amino acid modifications suchas amino acid substitution, insertion, addition, or deletion.

In one embodiment, the Fc region variants of Disclosure B or antibodiescomprising the Fc region variants can be assessed for their (human)FcRn-binding activity in a neutral pH range and/or in an acidic pH rangein the same manner as described above.

In one embodiment, a method for modifying antibody constant regions toproduce the Fc region variants of Disclosure B may be based, forexample, on assessment of several constant region isotypes (IgG1, IgG2,IgG3, and IgG4) to select isotypes that have a reduced antigen-bindingactivity in an acidic pH range and/or have an increased dissociationrate in an acidic pH range. An alternative method may be based onintroduction of amino acid substitutions into the amino acid sequence ofa native IgG isotype to reduce the antigen-binding activity in an acidicpH range (e.g., pH 5.8) and/or to increase the dissociation rate in anacidic pH range. The hinge region sequence of an antibody constantregion varies greatly across isotypes (IgG1, IgG2, IgG3, and IgG4), anddifferences in the hinge-region amino acid sequence can have asignificant impact on the antigen-binding activity. Therefore, isotypeswith reduced antigen-binding activity in an acidic pH range and/orincreased dissociation rate in an acidic pH range can be selected byselecting suitable isotypes depending on the type of antigen or epitope.Furthermore, since differences in the hinge-region amino acid sequencecan have a significant impact on the antigen-binding activity, aminoacid substitutions in the amino acid sequences of native isotypes can belocated in the hinge region.

In an alternative embodiment, Disclosure B provides a use of an antibodycontaining the above-described Fc region variant of Disclosure B toaccelerate the release of the antibody that has been internalized intocells in an antigen-bound form to the outside of the cells in anantigen-free form. Herein, “release of an antibody that has beeninternalized into cells in an antigen-bound form to the outside of thecells in an antigen-free form” does not necessarily mean that theantibody that has been internalized into cells in an antigen-bound formis completely released to the outside of the cells in an antigen-freeform. It is acceptable that the proportion of the antibody released inan antigen-free form to the outside of the cells is increased ascompared to that before modification of its FcRn-binding domain (forexample, before increasing the FcRn-binding activity of the antibody inan acidic pH range). It is preferable that the antibody released to theoutside of the cells maintains its antigen-binding activity.

The “ability to eliminate antigen from plasma” or an equivalent term canrefer to the ability to eliminate antigen from plasma when an antibodyis administered or secreted in vivo. Thus, “the antibody's ability toeliminate antigen from plasma is increased” can mean that when anantibody is administered, for example, the rate of antigen eliminationfrom plasma is increased as compared to that before modification of itsFcRn-binding domain. The increase in the antibody's activity of antigenelimination from plasma can be assessed, for example, by administering asoluble antigen and the antibody in vivo, and measuring theconcentration of the soluble antigen in plasma after administration. Thesoluble antigen may be an antibody-bound or antibody-free antigen, andtheir concentrations can be determined as “antibody-bound antigenconcentration in plasma” and “antibody-free antigen concentration inplasma”, respectively. The latter is synonymous with “free antigenconcentration in plasma”. The “total antigen concentration in plasma”can refer to the sum of antibody-bound antigen concentration andantibody-free antigen concentration.

In an alternative embodiment, Disclosure B provides a method forprolonging the plasma retention time of an antibody containing the Fcregion variant of Disclosure B. Native human IgG can bind to FcRnderived from nonhuman animals. For example, since native human IgG canbind to mouse FcRn more strongly than to human FcRn (Ober et al., Intl.Immunol. 13(12):1551-1559 (2001)), the antibodies can be administered tomice for assessing the properties of the antibodies. Alternatively, forexample, mice with their own FcRn gene has been disrupted but insteadhave and express the human FcRn gene as a transgene (Roopenian et al.,Meth. Mol. Biol. 602:93-104 (2010)) are also suitable for assessing theantibodies.

Within the scope of Disclosures A and B described herein, the plasmaconcentration of free antigen not bound to the antibody or the ratio offree antigen concentration to the total antigen concentration can bedetermined (e.g., Ng et al., Pharm. Res. 23(1):95-103 (2006)).Alternatively, when an antigen exhibits a particular function in vivo,whether the antigen is bound to an antibody that neutralizes the antigenfunction (antagonistic molecule) can be assessed by testing whether theantigen function is neutralized. Whether the antigen function isneutralized can be evaluated by measuring a particular in vivo markerreflective of the antigen function. Whether an antigen is bound to anantibody that activates the antigen function (agonistic molecule) can beassessed by measuring a particular in vivo marker reflective of theantigen function.

There are no particular limitations on measurements such asdetermination of the free antigen concentration in plasma, determinationof the ratio of the amount of free antigen in plasma to the amount oftotal antigen in plasma, and in vivo marker measurement; however, themeasurements can be preferably carried out after a certain period oftime following antibody administration. In the context of Disclosure B,“after a certain period of time following antibody administration” isnot particularly limited, and the period can be appropriately determinedby those of ordinary skill in the art depending on the properties of theadministered antibody and others, and includes, for example, one day,three days, seven days, 14 days, or 28 days after antibodyadministration. Herein, the term “plasma antigen concentration” canrefer to either “total antigen concentration in plasma” which is the sumof antibody-bound antigen concentration and antibody-free antigenconcentration, or “free antigen concentration in plasma” which isantibody-free antigen concentration.

Molar ratio of antigen to antibody can be calculated using the formula:C=A/B, wherein value A is the molar concentration of antigen at eachtime point, value B is the molar concentration of antibody at each timepoint, and value C is the molar concentration of antigen per molarconcentration of antibody (molar ratio of antigen/antibody) at each timepoint.

A smaller C value indicates a higher efficiency of antigen eliminationper antibody, and a larger C value indicates a lower efficiency ofantigen elimination per antibody.

In some aspects, when an antibody of Disclosure B is administered, themolar ratio of antigen/antibody is reduced by 2-fold, 5-fold, 10-fold,20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold or more,as compared to when a reference antibody containing a native human IgGFc region as a human FcRn-binding domain is administered.

The reduction of total antigen concentration in plasma or molar ratio ofantigen/antibody can be assessed using methods know in the art, such asthat described in Examples 6, 8, and 13 of WO2011/122011. Morespecifically, they can be assessed based on either an antigen-antibodyco-injection model or a steady-state antigen infusion model using thehuman FcRn transgenic mouse line 32 or 276 (Jackson Laboratories,Methods Mol. Biol. 602:93-104 (2010)), when an antibody of interest inDisclosure B does not cross-react with the mouse counterpart antigen.When the antibody cross-reacts with the mouse counterpart, it can beassessed by simply injecting the antibody into the human FcRn transgenicmouse line 32 or 276 (Jackson Laboratories). In the co-injection model,a mixture of the antibody and antigen is administered to mice. In thesteady-state antigen infusion model, an infusion pump filled with anantigen solution is implanted into mice to achieve a constant antigenconcentration in plasma, and then the antibody is injected into themice. All test antibodies are administered at the same dosage. The totalantigen concentration in plasma, free antigen concentration in plasma,and antibody concentration in plasma can be measured at appropriate timepoints.

To assess the long-term effects of an antibody of Disclosure B, thetotal or free antigen concentration in plasma, or the molar ratio ofantigen/antibody can be measured two days, four days, seven days, 14days, 28 days, 56 days, or 84 days after administration. In other words,for assessing properties of the antibody, an antigen concentration inplasma in a long period of time can be determined by measuring the totalor free antigen concentration in plasma, or the molar ratio ofantigen/antibody two days, four days, seven days, 14 days, 28 days, 56days, or 84 days after antibody administration. Whether the antigenconcentration in plasma or the molar ratio of antigen/antibody isreduced with the antibody can be determined by assessing such reductionsat one or more time points as described above.

To assess the short-term effects of an antibody of Disclosure B, thetotal or free antigen concentrations in plasma, or the molar ratio ofantigen/antibody can be measured 15 minutes, one hour, two hours, fourhours, eight hours, 12 hours, or 24 hours after administration. In otherwords, for assessing properties of the antibody, an antigenconcentration in plasma in a short period of time can be determined bymeasuring the total or free antigen concentrations in plasma, or themolar ratio of antigen/antibody 15 minutes, one hour, two hours, fourhours, eight hours, 12 hours, or 24 hours after administration. When theplasma retention in human is difficult to determine, it may be predictedbased on the plasma retention in mice (for example, normal mice, humanantigen-expressing transgenic mice, or human FcRn-expressing transgenicmice) or in monkeys (for example, cynomolgus monkeys).

In an alternative embodiment, Disclosure B relates to an antibodycomprising the Fc region variant of Disclosure B described above. Thevarious embodiments of the antibodies described within the scope ofDisclosures A and B described herein can be applicable without opposingthe common technical knowledge in the art and unless there areinconsistencies in the context.

In one embodiment, antibodies comprising an Fc region variant ofDisclosure B are useful as therapeutic antibodies for treating humanpatients with auto-immune diseases, transplantation rejection (graftversus host disease), other inflammatory diseases, or allergy diseases,as described in WO2013/046704.

In one embodiment, antibodies comprising an Fc region variant ofDisclosure B may have modified sugar chains. Antibodies with modifiedsugar chains can include, for example, antibodies with modifiedglycosylation (WO99/54342), antibodies that are deficient in fucose(WO00/61739, WO02/31140, WO2006/067847, WO2006/067913), and antibodieshaving sugar chains with bisecting GlcNAc (WO02/79255). In oneembodiment, the antibodies may be deglycosylated. In some embodiments,the antibodies comprise, for example, mutations at the heavy-chainglycosylation site to inhibit glycosylation at such location asdescribed in WO2005/03175. Such non-glycosylated antibodies can beprepared by modifying the heavy-chain glycosylation site, i.e., byintroducing the N297Q or N297A substitution according to EU numbering,and expressing the proteins in appropriate host cells.

In an alternative embodiment, Disclosure B relates to a composition or apharmaceutical composition comprising an antibody containing such an Fcregion variant. The various embodiments of the compositions orpharmaceutical compositions described within the scope of Disclosures Aand B herein can be applicable without opposing the common technologicalknowledge in the art and unless there are inconsistencies in thecontext. Such compositions can be used for enhancing the plasmaretention (in subjects, when an antibody of the Disclosure B isadministered (applied) to the subjects).

In an alternative embodiment, Disclosure B relates to nucleic acidsencoding an Fc region variant or antibodies containing the Fc regionvariant. The various embodiments of the nucleic acids described withinthe scope of Disclosures A and B described herein can be applicablewithout opposing the common technical knowledge in the art and unlessthere are inconsistencies in the context. Alternatively, Disclosure Brelates to vectors comprising the nucleic acids. The various embodimentsthereof within the scope of Disclosures A and B described herein can beapplicable without opposing the common technical knowledge in the artand unless there are inconsistencies in the context. Alternatively,Disclosure B relates to hosts or host cells comprising the vectors. Thevarious embodiments thereof within the scope of Disclosures A and Bdescribed herein can be applicable without opposing the common technicalknowledge in the art and unless there are inconsistencies in thecontext.

In an alternative embodiment, Disclosure B relates to methods forproducing an Fc region variant comprising an FcRn-binding domain or anantibody comprising the Fc region variant, which comprise culturing thehost cells described above, or growing the hosts described above andcollecting the Fc region variant or antibody comprising the Fc regionvariant from the cell culture, materials secreted from the hosts. Inthis case, Disclosure B may include production methods optionallyfurther comprising any one or more of: (a) selecting an Fc regionvariant with enhanced FcRn-binding activity under an acidic pH conditionas compared to that of an Fc region of a native IgG; (b) selecting an Fcregion variant whose binding activity to a (pre-existing) ADA is notsignificantly enhanced under a neutral pH condition as compared to thatof an Fc region of a native IgG; (c) selecting an Fc region variant withincreased plasma retention as compared to that of an Fc region of anative IgG; and (d) selecting an antibody comprising an Fc regionvariant that can promote elimination of an antigen from plasma ascompared to a reference antibody comprising an Fc region of a nativeIgG.

From the perspective of assessing the plasma retention of an Fc regionvariant of Disclosure B, without limitations, it can be preferable thatan antibody comprising the Fc region variant produced in Disclosure Band the “reference antibody comprising the Fc region of a native IgG”are identical to each other except for the Fc region to be compared. TheFcRn can be human FcRn.

For example, after producing an antibody comprising an Fc region variantof Disclosure B, the antibody may be compared with the referenceantibody comprising a native IgG Fc region in terms of the FcRn-bindingactivity under an acidic pH condition (e.g., pH 5.8) using BIACORE® orother known techniques, to select an Fc region variant or an antibodycomprising the Fc region variant whose FcRn-binding activity has beenincreased under the acidic pH condition.

Alternatively, for example, after producing an antibody comprising an Fcregion variant of Disclosure B, the antibody may be compared with areference antibody comprising a native IgG Fc region in terms of theADA-binding activity under a neutral pH condition byelectrochemiluminescence (ECL) or known techniques, to select an Fcregion variant or antibodies comprising the Fc region variant whoseADA-binding activity has not been significantly increased under theneutral pH condition.

Alternatively, for example, after producing an antibody comprising an Fcregion variant of Disclosure B, the antibody may be compared with areference antibody comprising a native IgG Fc region by conductingantibody pharmacokinetic tests using plasma, for example, from mice,rats, rabbits, dogs, monkeys, or humans, to select Fc region variants orantibodies comprising the Fc region variant which are demonstrated tohave improved plasma retention in the subjects.

Alternatively, for example, after producing an antibody comprising an Fcregion variant of Disclosure B, the antibody may be compared with areference antibody comprising a native IgG Fc region by conductingantibody pharmacokinetic tests using plasma, for example, from mice,rats, rabbits, dogs, monkeys, or humans, to select Fc region variants orantibodies comprising the Fc region variant which have enhanced antigenelimination from plasma.

Alternatively, for example, the selection methods described above may beappropriately combined, if needed.

In one embodiment, Disclosure B relates to a method for producing an Fcregion variant comprising an FcRn-binding domain or an antibodycomprising the variant, wherein the method comprises substituting aminoacids in a way such that the resulting Fc region variant or the antibodycomprising the variant comprises Ala at position 434; Glu, Arg, Ser, orLys at position 438; and Glu, Asp, or Gln at position 440, according toEU numbering. In an additional embodiment, such method comprisessubstituting the amino acids in a way such that the resulting Fc regionvariant or the antibody comprising the variant further comprises, Ile orLeu at position 428 and/or Ile, Leu, Val, Thr, or Phe at position 436,according to EU numbering. In a further embodiment, the amino acids aresubstituted in a way such that the resulting Fc region variant or theantibody comprising the variant produced according to the method furthercomprises Leu at position 428 and/or Val or Thr at position 436,according to EU numbering.

In one embodiment, Disclosure B relates to a method for producing an Fcregion variant comprising an FcRn-binding domain or an antibodycomprising the variant, wherein the method comprises substituting aminoacids in a way such that the resulting Fc region variant or the antibodycomprising the variant comprises Ala at position 434; Arg or Lys atposition 438; and Glu or Asp at position 440, according to EU numbering.In an additional embodiment, such method comprises substituting theamino acids in a way such that the resulting Fc region variant or theantibody comprising the variant further comprises, Ile or Leu atposition 428 and/or Ile, Leu, Val, Thr, or Phe at position 436,according to EU numbering. In a further embodiment, the amino acids aresubstituted in a way such that the resulting Fc region variant or theantibody comprising the variant produced according to the method furthercomprises Leu at position 428 and/or Val or Thr at position 436,according to EU numbering.

In one embodiment, such method comprises substituting all amino acids atpositions 434, 438, and 440 with Ala; Glu, Arg, Ser, or Lys; and Glu,Asp, or Gln, respectively. In an additional embodiment, such methodcomprises substituting the amino acids in a way such that the resultingFc region variant or the antibody comprising the variant furthercomprises, Ile or Leu at position 428 and/or Ile, Leu, Val, Thr, or Pheat position 436, according to EU numbering. In a further embodiment, theamino acids are substituted in a way such that the resulting Fc regionvariant or the antibody comprising the variant produced according to themethod further comprises Leu at position 428 and/or Val or Thr atposition 436, according to EU numbering.

In an alternative embodiment, Disclosure B relates to an Fc regionvariant or an antibody comprising the Fc region variant obtained by anyof the production methods of Disclosure B described above.

In an alternative embodiment, Disclosure B provides methods for reducingthe (pre-existing) ADA-binding activity of antibodies comprising an Fcregion variant with increased FcRn-binding activity at an acidic pH; andmethods for producing Fc region variants with increased FcRn-bindingactivity at an acidic pH (e.g., pH 5.8) and reduced pre-existingADA-binding activity, which comprise: (a) providing an antibodycomprising an Fc region (variant) whose FcRn-binding activity at anacidic pH has been increased as compared to a reference antibody; and(b) introducing into the Fc region, according to EU numbering, (i) anamino acid substitution with Ala at position 434; (ii) an amino acidsubstitution with any one of Glu, Arg, Ser, and Lys at position 438; and(iii) an amino acid substitution with any one of Glu, Asp, and Gln atposition 440, (iv) optionally, an amino acid substitution with Ile orLeu at position 428; and/or (v) optionally, an amino acid substitutionwith any one of Ile, Leu, Val, Thr, and Phe at position 436.

In some embodiments, the Fc domain (variant) in step (a) is preferably ahuman IgG Fc domain (variant). Furthermore, to increase the FcRn-bindingactivity at an acidic pH and to decrease the (pre-existing) ADA-bindingactivity in a neutral pH range (e.g., pH 7.4), the Fc region (variant)is to contain a combination of substituted amino acids selected from thegroup consisting of: (a) N434A/Q438R/S440E; (b) N434A/Q438R/S440D; (c)N434A/Q438K/S440E; (d) N434A/Q438K/S440D; (e) N434A/Y436T/Q438R/S440E;(f) N434A/Y436T/Q438R/S440D; (g) N434A/Y436T/Q438K/S440E; (h)N434A/Y436T/Q438K/S440D; (i) N434A/Y436V/Q438R/S440E; (j)N434A/Y436V/Q438R/S440D; (k) N434A/Y436V/Q438K/S440E; (l)N434A/Y436V/Q438K/S440D; (m) N434A/R435H/F436T/Q438R/S440E; (n)N434A/R435H/F436T/Q438R/S440D; (o) N434A/R435H/F436T/Q438K/S440E; (p)N434A/R435H/F436T/Q438K/S440D; (q) N434A/R435H/F436V/Q438R/S440E; (r)N434A/R435H/F436V/Q438R/S440D; (s) N434A/R435H/F436V/Q438K/S440E; (t)N434A/R435H/F436V/Q438K/S440D; (u) M428L/N434A/Q438R/S440E; (v)M428L/N434A/Q438R/S440D; (w) M428L/N434A/Q438K/S440E; (x)M428L/N434A/Q438K/S440D; (y) M428L/N434A/Y436T/Q438R/S440E; (z)M428L/N434A/Y436T/Q438R/S440D; (aa) M428L/N434A/Y436T/Q438K/S440E; (ab)M428L/N434A/Y436T/Q438K/S440D; (ac) M428L/N434A/Y436V/Q438R/S440E; (ad)M428L/N434A/Y436V/Q438R/S440D; (ae) M428L/N434A/Y436V/Q438K/S440E; (af)M428L/N434A/Y436V/Q438K/S440D; (ag)L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and (ah)L235R/G236R/A327G/A330S/P331 S/M428L/N434A/Y436T/Q438R/S440E.

The methods may optionally further comprise: (c) assessing whether the(pre-existing) ADA-binding activity of an antibody comprising a producedFc region variant is reduced as compared to the binding activity of thereference antibody.

Alternatively, the methods may be used as a method for enhancing therelease of an antibody that has been internalized into cells in anantigen-bound form to the outside of the cells in an antigen-free form,without significantly increasing the (pre-existing) ADA-binding activityof the antibody at a neutral pH.

Disclosure C

Disclosure C also relates to anti-IL-8 antibodies, nucleic acidsencoding the antibodies, pharmaceutical compositions comprising theantibodies, methods for producing the antibodies, and uses of theantibodies in treating diseases related to IL-8, as described in detailhereinbelow. The meanings of the terms given hereinbelow applythroughout the description of Disclosure C herein, without beingcontrary to the common technical knowledge of those of ordinary skill inthe art as well as embodiments known to those or ordinary skill in theart.

I. Definitions within the Scope of Disclosure C

Within the scope of Disclosure C described herein, “acidic pH” refers topH that may be selected, for example, from pH 4.0 to pH 6.5. In oneembodiment, acidic pH refers to, but is not limited to, pH 4.0, pH 4.1,pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0,pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9,pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, or pH 6.5. In a specificembodiment, the term acidic pH refers to the pH 5.8.

Within the scope of Disclosure C described herein, “neutral pH” refersto pH selected, for example, from 6.7 to pH 10.0. In one embodiment,neutral pH refers to, but is not limited to, pH 6.7, pH 6.8, pH 6.9, pH7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH8.8, pH 8.9, pH 9.0, pH 9.5, or pH 10.0. In a specific embodiment, theterm neutral pH refers to the pH 7.4.

The term “IL-8”, as used in Disclosure C, refers to any native IL-8derived from any vertebrates, primates (e.g., humans, cynomolgusmonkeys, rhesus monkeys) and other mammals (e.g., dogs and rabbits),unless otherwise indicated. The term “IL-8” encompasses full-lengthIL-8, unprocessed IL-8 as well as any form of IL-8 that results fromprocessing in the cell. The term “IL-8” also encompasses derivatives ofnative IL-8, for example, splice variants or allelic variants. The aminoacid sequence of an exemplary human IL-8 is shown in SEQ ID NO:66.

The terms “anti-IL-8 antibody” and “an antibody that binds to IL-8”refer to an antibody that is capable of binding IL-8 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting IL-8.

In one embodiment, the extent of binding of an anti-IL-8 antibody to anunrelated, non-IL-8 protein is, for example, less than about 10% of thebinding of the antibody to IL-8.

“Affinity” within the scope of the description of Disclosure C hereingenerally refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used within the scope of the description of Disclosure Cherein, “binding affinity” refers to intrinsic binding affinity whichreflects a 1:1 interaction between members of a binding pair (e.g.,antibody and antigen). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (KD). Bindingaffinity can be measured using methods known in the art, including thosedescribed within the scope of the description of Disclosure C herein.

In certain embodiments, an antibody that binds to IL-8 may have adissociation constant (KD) of, for example, ≤1000 nM, ≤100 nM, ≤10 nM,≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less, from 10⁻⁸ Mto 10⁻¹³M, from 10⁻⁹M to 10⁻¹³ M).

The term “antibody” within the scope of the description of Disclosure Cherein is used in the broadest sense and includes, but is not limitedto, monoclonal antibodies, polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they exhibit the desired antigen-binding activity.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen by, for example, 50%, 60%, 70%, or 80% or more; andconversely, the reference antibody blocks binding of the antibody to itsantigen by, for example, 50%, 60%, 70%, or 80% or more. Here, anexemplary competition assay can be used without being limited thereto.

A “chimeric antibody” refers to an antibody in which a portion of theheavy and/or light chain is derived from a particular source or species,while the remaining portion is derived from a different source orspecies.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody may comprisesubstantially at least one, and typically two, variable regions, inwhich all (or substantially all) of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all (or substantially all) of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody.

The term “monoclonal antibody” as used within the scope of thedescription of Disclosure C herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies that constitute the population are identical and/or bind thesame epitope, except for possible variant antibodies, for example, thosecontaining naturally occurring mutations or arising during production ofa monoclonal antibody preparation, which are generally present in minoramounts. In contrast to polyclonal antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody in a monoclonal antibodypreparation is directed against a single determinant on an antigen.Thus, the modifier “monoclonal” indicates the characteristics of anantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any specific method. For example, the monoclonal antibodiesto be used in accordance with Disclosure C may be made by varioustechniques, including but not limited to the hybridoma method,recombinant DNA methods, phage-display methods, and methods utilizingtransgenic animals comprising all or part of the human immunoglobulinloci, such methods and other exemplary methods for making monoclonalantibodies being described herein.

Within the scope of Disclosure C described herein, “native antibody”refers to immunoglobulin molecules with various naturally occurringstructures. In an embodiment, a native IgG antibody, for example, is aheterotetrameric glycoprotein of about 150,000 daltons composed of twoidentical light chains and two identical heavy chains that aredisulfide-bonded. In the order from N- to C-terminus, each heavy chainhas a variable region (VH), which is also referred to as a variableheavy-chain domain or heavy-chain variable domain, followed by threeconstant domains (CH1, CH2, and CH3). Likewise, in the order from N- toC-terminus, each light chain has a variable region (VL), which is alsoreferred to as a variable light-chain domain or light-chain variabledomain, followed by a constant light-chain (CL) domain. An antibodylight chain may be assigned to one of the two types, called kappa (K)and lambda (λ), based on the amino acid sequence of its constant domain.Such constant domains for use in the Disclosure C include those of anyreported allotype (allele) or any subclass/isotype. The heavy-chainconstant region includes, but is not limited to, the constant region ofa native IgG antibody (IgG1, IgG2, IgG3, and IgG4). Known IgG1 allelesinclude, for example, IGHG1*01, IGHG1*02, IGHG1*03, IGHG1*04, andIGHG1*05 (see at imgt.org), and any of these can be used as a nativehuman IgG1 sequence. The constant domain sequence may be derived from asingle allele or subclass/isotype, or from multiple alleles orsubclasses/isotypes. Specifically, such antibodies include, but are notlimited to, an antibody whose CH1 is derived from IGHG1*01 and CH2 andCH3 are derived from IGHG1*02 and IGHG1*01, respectively.

“Effector functions” within the scope of the description of Disclosure Cherein refers to biological activities attributable to the Fc region ofan antibody, which may vary with the antibody isotype. Examples ofantibody effector functions include: C1q binding andcomplement-dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation, but are not limited thereto.

The term “Fc region” within the scope of the description of Disclosure Cherein is used to define a C-terminal region of an immunoglobulin heavychain that contains at least a portion of the constant region. The termincludes native Fc regions and variant Fc regions. The native Fc regionindicates the Fc region of a native antibody.

In one embodiment, a human IgG heavy-chain Fc region extends from theamino acid residue of Cys226 or Pro230 to the carboxyl terminus of theheavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine(residues 446-447) of the Fc region may or may not be present. Unlessotherwise specified within the scope of the description of Disclosure Cherein, the numbering of amino acid residues in the Fc region orconstant region is according to the EU numbering system, also called theEU index, as described in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991.

“Framework” or “FR” within the scope of the description of Disclosure Cherein refers to variable domain residues other than hypervariableregion (HVR) residues. The FR of a variable domain generally consists offour FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FRsequences generally appear in the following sequence:FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4 in VH (or VL).

A “human consensus framework” within the scope of the description ofDisclosure C herein is a framework that represents the most commonlyoccurring amino acid residues in a selection of human immunoglobulin VLor VH framework sequences. Generally, the selection of humanimmunoglobulin VL or VH sequences is from a subgroup of variable domainsequences. Generally, the subgroup of sequences is a subgroup accordingto Kabat et al., Sequences of proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991).

In one embodiment, the subgroup for the VL is subgroup id as in Kabat etal., supra. In one embodiment, the subgroup for the VH is subgroup IIIas in Kabat et al., supra.

An “acceptor human framework” for purposes within the scope of thedescription of Disclosure C herein is a framework comprising the aminoacid sequence of a VL or VH framework derived from a humanimmunoglobulin framework or a human consensus framework. An acceptorhuman framework “derived from” a human immunoglobulin framework or ahuman consensus framework may comprise the same amino acid sequencethereof, or it may contain existing amino acid sequence substitutions.In some embodiments, the number of existing amino acid substitutions are10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less. In one embodiment, the VL acceptor humanframework is identical to the VL human immunoglobulin framework sequenceor human consensus framework sequence.

The term “variable region” or “variable domain” within the scope of thedescription of Disclosure C herein refers to the domain of an antibodyheavy or light chain involved in binding of the antibody to an antigen.The variable regions of the heavy chain and light chain (VH and VL,respectively) of a native antibody generally have similar structures,with each domain comprising four conserved framework regions (FRs) andthree hypervariable regions (HVRs). (See, Kindt et al., Kuby Immunology,6^(th) ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VLdomain is sufficient to confer antigen-binding specificity, but is notlimited thereto. Furthermore, antibodies that bind to a particularantigen may be isolated using a VH or VL domain from an antibody thatbinds the antigen to screen a library of complementary VL or VH domains,respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887(1993); Clarkson et al., Nature 352:624-628 (1991).

The term “hypervariable region” or “HVR” as used within the scope of thedescription of Disclosure C herein refers to each of the regions of anantibody variable domain which are hypervariable in sequence(“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six hypervariable regions: three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3).

Without being limited thereto, exemplary HVRs herein include: (a)hypervariable loops in which amino acid residues are 26-32 (L1), 50-52(L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia andLesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs in which amino acidresidues are 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65(H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)); (c) antigen contacts inwhich amino acid residues are 27c-36 (L1), 46-55 (L2), 89-96 (L3),30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol.Biol. 262:732-745 (1996)); and (d) combinations of (a), (b), and/or (c),including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2),49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102(H3).

Unless otherwise instructed, HVRs and other residues in variable regions(e.g., FR residues) are numbered as in Kabat et al., supra.

An “individual” within the scope of the description of Disclosure Cherein is a mammal. Mammals include, but are not limited to,domesticated animals (e.g., cows, sheep, cats, dogs, and horses),primates (e.g., humans and non-human primates such as monkeys), rabbits,and rodents (e.g., mice and rats). In certain embodiments, the“individual” is a human.

An “isolated” antibody within the scope of the description of DisclosureC herein is one which has been separated from a component of its naturalenvironment. In some embodiments, an antibody is purified to be greaterthan 95% or 99% in purity as determined, for example,electrophoretically (e.g., SDS-PAGE, isoelectric focusingelectrophoresis (IEF), capillary electrophoresis) or chromatographically(e.g., ion exchange or reverse phase HPLC). For review of methods forassessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr.B 848:79-87 (2007).

An “isolated” nucleic acid within the scope of the description ofDisclosure C herein refers to a nucleic acid molecule that has beenseparated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-IL-8 antibody” within the scopeof the description of Disclosure C herein refers to one or more nucleicacid molecules encoding anti-IL-8 antibody heavy and light chains (orfragments thereof), including such nucleic acid(s) in a single vector orseparate vectors, nucleic acid(s) present at one or more locations in ahost cell.

Within the scope of the description of Disclosure C herein, the terms“host cell,” “host cell line,” and “host cell culture” are usedinterchangeably and refer to cells into which an exogenous nucleic acidhas been introduced, including the progeny of such cells. Host cellsinclude “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. A progeny may not be completely identical in itsnucleic acid content to a parent cell, but may contain mutations. Amutant progeny that has the same function or biological activity as thatscreened or selected in the originally transformed cell are includedherein.

The term “vector”, as used within the scope of the description ofDisclosure C herein, refers to a nucleic acid molecule capable ofpropagating another nucleic acid to which it is linked. The termincludes vectors in a self-replicating nucleic acid structure as well asvectors introduced into a host cell and become incorporated into itsgenome. Certain vectors are capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “expression vectors.”

Within the scope of the description of Disclosure C herein, the term“package insert” is used to refer to instructions customarily includedin commercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, combinationtherapy, contraindications and/or warnings concerning the use of suchtherapeutic products.

Within the scope of the description of Disclosure C herein, “percent (%)amino acid sequence identity” with respect to a reference polypeptidesequence is defined as the percentage of amino acid residues in acandidate sequence that are identical to the amino acid residues in thereference polypeptide sequence after sequence alignment, by introducinggaps if necessary and not considering any conservative substitutions aspart of the sequence identity, in order to achieve the maximum percentsequence identity. Alignment for purposes of determining percent aminoacid sequence identity can be achieved in various ways within the scopeof the ability of those of ordinary skill in the art, for instance, byusing publicly available computer software such as BLAST, BLAST-2,ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Co., Ltd.).Those of ordinary skill in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, values of the % amino acid sequenceidentity are generated, for example, using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 was authored by Genentech, Inc.,and the source code has been filed with user documentation in the U.S.Copyright Office, Washington D.C., 20559, where it is registered underU.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX (registered trademark) operating system,including digital UNIX (registered trademark) V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to a given amino acid sequence B) iscalculated as follows: 100 times the fraction X/Y, where X is the numberof amino acid residues scored as identical matches in the programalignment of A and B by the sequence alignment program ALIGN-2, andwhere Y is the total number of amino acid residues in B. It will beappreciated that where the length of amino acid sequence A is not equalto the length of amino acid sequence B, the % amino acid sequenceidentity of A to B will not equal the % amino acid sequence identity ofB to A. Unless specifically stated otherwise, all % values of amino acidsequence identity are obtained using the ALIGN-2 computer program asdemonstrated under the scope of the description of Disclosure C herein.

Within the scope of Disclosure C described herein, a “pharmaceuticalcomposition” generally refers to an agent for treating, preventing,examining, or diagnosing diseases. A “pharmaceutically acceptablecarrier” refers to an ingredient in a pharmaceutical composition, otherthan an active ingredient, which is nontoxic to a subject. Suchpharmaceutically acceptable carriers include, but are not limited to,buffers, excipients, stabilizers, and preservatives.

As used within the scope of Disclosure C described herein, “treatment”(and grammatical variations thereof such as “treat” or “treating”)refers to a clinical intervention in an attempt to alter the naturalcourse of the individual being treated. Such a clinical intervention canbe performed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, prevention of the occurrence or recurrence of a disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, prevention of metastasis, decrease of therate of disease progression, amelioration or palliation of the diseasestate, and remission or improvement of prognosis. In one embodiment, anantibody of Disclosure C can be used to slow down the progression of adisease or disorder.

Within the scope of Disclosure C described herein, an “effective amount”of an antibody or pharmaceutical composition refers to an amount that iseffective when used at doses and for periods of time necessary toachieve the desired therapeutic or prophylactic result.

II. Compositions and Methods

In one embodiment, Disclosure C is based on the applicability ofanti-IL-8 antibodies that have pH-dependent affinity for IL-8 aspharmaceutical compositions. The antibodies of Disclosure C are useful,for example, in diagnosing or treating diseases where IL-8 is present inan excessive amount.

A. Exemplary Anti-IL-8 Antibodies

In one embodiment, Disclosure C provides an anti-IL-8 antibody havingpH-dependent affinity for IL-8.

In one embodiment, Disclosure C provides an anti-IL-8 antibody havingpH-dependent affinity for IL-8, which comprises a sequence with at leastone, two, three, four, five, six, seven, or eight amino acidsubstitution(s) within the amino acid sequences of: (a) HVR-H1 whichcomprises the amino acid sequence of SEQ ID NO:67; (b) HVR-H2 whichcomprises the amino acid sequence of SEQ ID NO:68; (c) HVR-H3 whichcomprises the amino acid sequence of SEQ ID NO:69; (d) HVR-L1 whichcomprises the amino acid sequence of SEQ ID NO:70; (e) HVR-L2 whichcomprises the amino acid sequence of SEQ ID NO:71; and (f) HVR-L3 whichcomprises the amino acid sequence of SEQ ID NO:72.

In another embodiment, Disclosure C provides an anti-IL-8 antibodyhaving pH-dependent affinity for IL-8, which comprises at least oneamino acid substitution(s) in at least one of the amino acid sequencesof: (a) HVR-H1 which comprises the amino acid sequence of SEQ ID NO:67;(b) HVR-H2 which comprises the amino acid sequence of SEQ ID NO:68; (c)HVR-H3 which comprises the amino acid sequence of SEQ ID NO:69; (d)HVR-L1 which comprises the amino acid sequence of SEQ ID NO:70; (e)HVR-L2 which comprises the amino acid sequence of SEQ ID NO:71; and (f)HVR-L3 which comprises the amino acid sequence of SEQ ID NO:72.

Unless otherwise specified, the amino acids may be substituted with anyother amino acid. In one embodiment, an anti-IL-8 antibody of DisclosureC comprises one or more amino acid substitution(s) at position(s)selected from the group consisting of: aspartic acid at position 1 inthe sequence of SEQ ID NO:67; (b) tyrosine at position 2 in the sequenceof SEQ ID NO:67; (c) tyrosine at position 3 in the sequence of SEQ IDNO:67; (d) leucine at position 4 in the sequence of SEQ ID NO:67; (e)serine at position 5 in the sequence of SEQ ID NO:67; (f) leucine atposition 1 in the sequence of SEQ ID NO:68; (g) isoleucine at position 2in the sequence of SEQ ID NO:68; (h) arginine at position 3 in thesequence of SEQ ID NO:68; (i) asparagine at position 4 in the sequenceof SEQ ID NO:68; (j) lysine at position 5 in the sequence of SEQ IDNO:68; (k) alanine at position 6 in the sequence of SEQ ID NO:68; (l)asparagine at position 7 in the sequence of SEQ ID NO:68; (m) glycine atposition 8 in the sequence of SEQ ID NO:68; (n) tyrosine at position 9in the sequence of SEQ ID NO:68; (o) threonine at position 10 in thesequence of SEQ ID NO:68; (p) arginine at position 11 in the sequence ofSEQ ID NO:68; (q) glutamic acid at position 12 in the sequence of SEQ IDNO:68; (r) tyrosine at position 13 in the sequence of SEQ ID NO:68; (s)serine at position 14 in the sequence of SEQ ID NO:68; (t) alanine atposition 15 in the sequence of SEQ ID NO:68; (u) serine at position 16in the sequence of SEQ ID NO:68; (v) valine at position 17 in thesequence of SEQ ID NO:68; (w) lysine at position 18 in the sequence ofSEQ ID NO:68; (x) glycine at position 19 in the sequence of SEQ IDNO:68; (y) glutamic acid at position 1 in the sequence of SEQ ID NO:69;(z) asparagine at position 2 in the sequence of SEQ ID NO:69; (aa)tyrosine at position 3 in the sequence of SEQ ID NO:69; (ab) arginine atposition 4 in the sequence of SEQ ID NO:69; (ac) tyrosine at position 5in the sequence of SEQ ID NO:69; (ad) aspartic acid at position 6 in thesequence of SEQ ID NO:69; (ae) valine at position 7 in the sequence ofSEQ ID NO:69; (af) glutamic acid at position 8 in the sequence of SEQ IDNO:69; (ag) leucine at position 9 in the sequence of SEQ ID NO:69; (ah)alanine at position 10 in the sequence of SEQ ID NO:69; (ai) tyrosine atposition 11 in the sequence of SEQ ID NO:69; (aj) arginine at position 1in the sequence of SEQ ID NO:70; (ak) alanine at position 2 in thesequence of SEQ ID NO:70; (al) serine at position 3 in the sequence ofSEQ ID NO:70; (am) glutamic acid at position 4 in the sequence of SEQ IDNO:70; (an) isoleucine at position 5 in the sequence of SEQ ID NO:70;(ao) isoleucine at position 6 in the sequence of SEQ ID NO:70; (ap)tyrosine at position 7 in the sequence of SEQ ID NO:70; (aq) serine atposition 8 in the sequence of SEQ ID NO:70; (ar) tyrosine at position 9in the sequence of SEQ ID NO:70; (as) leucine at position 10 in thesequence of SEQ ID NO:70; (at) alanine at position 11 in the sequence ofSEQ ID NO:70; (au) asparagine at position 1 in the sequence of SEQ IDNO:71; (av) alanine at position 2 in the sequence of SEQ ID NO:71; (aw)lysine at position 3 in the sequence of SEQ ID NO:71; (ax) threonine atposition 4 in the sequence of SEQ ID NO:71; (ay) leucine at position 5in the sequence of SEQ ID NO:71; (az) alanine at position 6 in thesequence of SEQ ID NO:71; (ba) aspartic acid at position 7 in thesequence of SEQ ID NO:71; (bb) glutamine at position 1 in the sequenceof SEQ ID NO:72; (bc) histidine at position 2 in the sequence of SEQ IDNO:72; (bd) histidine at position 3 in the sequence of SEQ ID NO:72;(be) phenylalanine at position 4 in the sequence of SEQ ID NO:72; (bf)glycine at position 5 in the sequence of SEQ ID NO:72; (bg)phenylalanine of position 6 in the sequence of SEQ ID NO:72; (bh)proline at position 7 in the sequence of SEQ ID NO:72; (bi) arginine atposition 8 in the sequence of SEQ ID NO:72; and (bj) threonine atposition 9 in the sequence of SEQ ID NO:72.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises oneor more amino acid substitution(s) at position(s) selected from thegroup consisting of: (a) alanine at position 6 in the sequence of SEQ IDNO:68; (b) glycine at position 8 in the sequence of SEQ ID NO:68; (c)tyrosine at position 9 in the sequence of SEQ ID NO:68; (d) arginine atposition 11 in the sequence of SEQ ID NO:68; and (e) tyrosine atposition 3 in the sequence of SEQ ID NO:69.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprisescombination(s) of amino acid substitutions at positions selected fromthe group consisting of: (a) alanine at position 6 in the sequence ofSEQ ID NO:68; (b) glycine at position 8 in the sequence of SEQ ID NO:68;(c) tyrosine at position 9 in the sequence of SEQ ID NO:68; (d) arginineat position 11 in the sequence of SEQ ID NO:68; and (e) tyrosine atposition 3 in the sequence of SEQ ID NO:69.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises aminoacid substitutions at the following positions: (a) tyrosine at position9 in the sequence of SEQ ID NO:68; (b) arginine at position 11 in thesequence of SEQ ID NO:68; and (c) tyrosine at position 3 in the sequenceof SEQ ID NO:69.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises aminoacid substitutions at the following positions: (a) alanine at position 6in the sequence of SEQ ID NO:68; (b) glycine at position 8 in thesequence of SEQ ID NO:68; (d) tyrosine at position 9 in the sequence ofSEQ ID NO:68; (e) arginine at position 11 in the sequence of SEQ IDNO:68; and (f) tyrosine at position 3 in the sequence of SEQ ID NO:69.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a)substitution of alanine with aspartic acid at position 6 in the sequenceof SEQ ID NO:68; (b) substitution of arginine with proline at position11 in the sequence of SEQ ID NO:68; and (c) substitution of tyrosinewith histidine at position 3 in the sequence of SEQ ID NO:69.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a)substitution of glycine with tyrosine at position 8 in the sequence ofSEQ ID NO:68; and (b) substitution of tyrosine with histidine atposition 9 in the sequence of SEQ ID NO:68.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a)substitution of alanine with aspartic acid at position 6 in the sequenceof SEQ ID NO:68; (b) substitution of glycine with tyrosine at position 8in the sequence of SEQ ID NO:68; (c) substitution of tyrosine withhistidine at position 9 in the sequence of SEQ ID NO:68; (d)substitution of arginine with proline at position 11 in the sequence ofSEQ ID NO:68; and (e) substitution of tyrosine with histidine atposition 3 in the sequence of SEQ ID NO:69.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprisesHVR-H2 which comprises the amino acid sequence of SEQ ID NO:73.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprisesHVR-H3 which comprises the amino acid sequence of SEQ ID NO:74.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprisesHVR-H1 comprising the amino acid sequence of SEQ ID NO:67, HVR-H2comprising the amino acid sequence of SEQ ID NO:73, and HVR-H3comprising the amino acid sequence of SEQ ID NO:74.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises oneor more amino acid substitution(s) at position(s) selected from thegroup consisting of: (a) serine at position 8 in the sequence of SEQ IDNO:70; (b) asparagine at position 1 in the sequence of SEQ ID NO:71; (c)leucine at position 5 in the sequence of SEQ ID NO:71; and (d) glutamineat position 1 in the sequence of SEQ ID NO:72. In a further embodiment,the anti-IL-8 antibody comprises a combination of any 2, 3, or all 4 ofthese substitutions.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprisescombination(s) of amino acid substitutions at positions selected fromthe group consisting of: (a) serine at position 8 in the sequence of SEQID NO:70; (b) asparagine at position 1 in the sequence of SEQ ID NO:71;(c) leucine at position 5 in the sequence of SEQ ID NO:71; and (d)glutamine at position 1 in the sequence of SEQ ID NO:72. In a furtherembodiment, the anti-IL-8 antibody comprises a combination of any 2, 3,or all 4 of these substitutions.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises aminoacid substitutions at the following positions: (a) asparagine atposition 1 in the sequence of SEQ ID NO:71; (b) leucine at position 5 inthe sequence of SEQ ID NO:71; and (c) glutamine at position 1 in thesequence of SEQ ID NO:72.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises aminoacid substitutions at the following positions: (a) serine at position 8in the sequence of SEQ ID NO:70; (b) asparagine at position 1 in thesequence of SEQ ID NO:71; (c) leucine at position 5 in the sequence ofSEQ ID NO:71; and (d) glutamine at position 1 in the sequence of SEQ IDNO:72.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a)substitution of asparagine with lysine at position 1 in the sequence ofSEQ ID NO:71; (b) substitution of leucine with histidine at position 5in the sequence of SEQ ID NO:71; and (c) substitution of glutamine withlysine at position 1 in the sequence of SEQ ID NO:72.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a)substitution of serine with glutamic acid at position 8 in the sequenceof SEQ ID NO:70; (b) substitution of asparagine with lysine at position1 in the sequence of SEQ ID NO:71; (c) substitution of leucine withhistidine at position 5 in the sequence of SEQ ID NO:71; and (c)substitution of glutamine with lysine at position 1 in the sequence ofSEQ ID NO:72.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprisesHVR-L2 comprising the amino acid sequence of SEQ ID NO:75.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprisesHVR-L3 comprising the amino acid sequence of SEQ ID NO:76.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprisesHVR-L1 comprising the amino acid sequence of SEQ ID NO:70, HVR-L2comprising the amino acid sequence of SEQ ID NO:75, and HVR-L3comprising the amino acid sequence of SEQ ID NO:76.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises aminoacid substitutions at the following positions: (a) alanine at position55 in the sequence of SEQ ID NO:77; (b) glycine at position 57 in thesequence of SEQ ID NO:77; (c) tyrosine at position 58 in the sequence ofSEQ ID NO:77; (d) arginine at position 60 in the sequence of SEQ IDNO:77; (e) glutamine at position 84 in the sequence of SEQ ID NO:77; (f)serine at position 87 in the sequence of SEQ ID NO:77; and (g) tyrosineat position 103 in the sequence of SEQ ID NO:77.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a)substitution of alanine with aspartic acid at position 55 in thesequence of SEQ ID NO:77; (b) substitution of glycine with tyrosine atposition 57 in the sequence of SEQ ID NO:77; (c) substitution oftyrosine with histidine at position 58 in the sequence of SEQ ID NO:77;(d) substitution of arginine with proline at position 60 in the sequenceof SEQ ID NO:77; (e) substitution of glutamine with threonine atposition 84 in the sequence of SEQ ID NO:77; (f) substitution of serinewith aspartic acid at position 87 in the sequence of SEQ ID NO:77; (g)and substitution of tyrosine with histidine at position 103 in thesequence of SEQ ID NO:77.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:78.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises alight chain variable region comprising the amino acid sequence of SEQ IDNO:79.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:78 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:79. The anti-IL-8 antibody that comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:78and a light chain variable region comprising the amino acid sequence ofSEQ ID NO:79 may be an anti-IL-8 antibody that binds to IL-8 in apH-dependent manner. The anti-IL-8 antibody that comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:78 and alight chain variable region comprising the amino acid sequence of SEQ IDNO:79 may be an anti-IL-8 antibody that maintains the IL-8-neutralizingactivity stably in vivo (for example, in plasma). The anti-IL-8 antibodythat comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:78 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:79 may be an antibody with lowimmunogenicity.

In an alternative aspect, anti-IL-8 antibodies of Disclosure C alsoinclude those that have pH-dependent affinity for IL-8 and contain atleast one amino acid substitution in at least any one amino acidsequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:102; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:103;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:104; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:105; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:106; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:107.

In an alternative aspect, anti-IL-8 antibodies of Disclosure C alsoinclude those that have pH-dependent affinity for IL-8 and contain atleast one amino acid substitution in an amino acid sequence of: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:108; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:109; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:110; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:111; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:112; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:113.

In an alternative aspect, anti-IL-8 antibodies of Disclosure C alsoinclude those that have pH-dependent affinity for IL-8 and contain atleast one amino acid substitution in at least any one amino acidsequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:114; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:115;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:116; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:117; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:118; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:119.

In an alternative aspect, anti-IL-8 antibodies of Disclosure C alsoinclude those that have pH-dependent affinity for IL-8 and contain atleast one amino acid substitution in at least any one amino acidsequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:120; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:121;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:122; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:123; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:124; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:125.

In an alternative aspect, anti-IL-8 antibodies of Disclosure C alsoinclude those that have pH-dependent affinity for IL-8 and contain atleast one amino acid substitution in at least any one amino acidsequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:126; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:127;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:128; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:129; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:130; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:131.

In an alternative aspect, anti-IL-8 antibodies of Disclosure C alsoinclude those that have pH-dependent affinity for IL-8 and contain atleast one amino acid substitution in at least any one amino acidsequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:132; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:133;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:134; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:135; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:136; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:137.

In an alternative aspect, anti-IL-8 antibodies of Disclosure C alsoinclude those that have pH-dependent affinity for IL-8 and contain atleast one amino acid substitution in at least any one amino acidsequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:138; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:139;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:140; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:141; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:142; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:143.

In one embodiment, an anti-IL-8 antibody of Disclosure C hasIL-8-neutralizing activity. The IL-8-neutralizing activity refers to anactivity of inhibiting the biological activity of IL-8, or may refer toan activity of inhibiting the receptor binding of IL-8.

In alternative aspect, an anti-IL-8 antibody of Disclosure C is ananti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. In thecontext of Disclosure C, an anti-IL-8 antibody that binds to IL-8 in apH-dependent manner refers to an antibody whose binding affinity forIL-8 at an acidic pH has been reduced as compared to the bindingaffinity for IL-8 at a neutral pH. For example, pH-dependent anti-IL-8antibodies include antibodies that have a higher affinity for IL-8 at aneutral pH than at an acidic pH. In one embodiment, an anti-IL-8antibody of Disclosure C has an IL-8 affinity at a neutral pH that is atleast 2 times, 3 times, 5 times, 10 times, 15 times, 20 times, 25 times,30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100times, 200 times, 400 times, 1000 times, 10000 times or more greaterthan the affinity at an acidic pH. The binding affinity can be measuredusing, without particular limitations, surface plasmon resonance methods(such as BIACORE®). The association rate constant (kon) and dissociationrate constant (koff) can be calculated using the BIACORE® T200Evaluation Software (GE Healthcare) based on a simple one-to-oneLangmuir binding model by fitting the association and dissociationsensorgrams simultaneously. The equilibrium dissociation constant (KD)is calculated as a ratio of koff/kon. To screen for antibodies whosebinding affinity varies depending on pH, without particular limitations,ELISA, kinetic exclusion assay (KinExA™), and others as well as surfaceplasmon resonance methods (such as BIACORE®) can be used. ThepH-dependent IL-8-binding ability refers to the property to bind IL-8 ina pH-dependent manner. Meanwhile, whether an antibody is capable ofbinding to IL-8 multiple times can be assessed by the methods describedin WO2009/125825.

In one embodiment, it is preferable that an anti-IL-8 antibody ofDisclosure C has a small dissociation constant (KD) for IL-8 at aneutral pH. In one embodiment, the dissociation constant of an antibodyof Disclosure C for IL-8 at a neutral pH is, for example, 0.3 nM orless, but is not limited thereto. In one embodiment, the dissociationconstant of an antibody of Disclosure C for IL-8 at a neutral pH is, forexample, 0.1 nM or less, but is not limited thereto. In one embodiment,the dissociation constant of an antibody of Disclosure C for IL-8 at aneutral pH is, for example, 0.03 nM or less, but is not limited thereto.

In one embodiment, it is preferable that an anti-IL-8 antibody ofDisclosure C has a small dissociation constant (KD) for IL-8 at pH 7.4.In one embodiment, the dissociation constant of an antibody ofDisclosure C for IL-8 at pH 7.4 is, for example, 0.3 nM or less, but isnot limited thereto. In one embodiment, the dissociation constant of anantibody of Disclosure C for IL-8 at pH 7.4 is, for example, 0.1 nM orless, but is not limited thereto. In one embodiment, the dissociationconstant of an antibody of Disclosure C for IL-8 at pH 7.4 is, forexample, 0.03 nM or less, but is not limited thereto.

In one embodiment, it is preferable that an anti-IL-8 antibody ofDisclosure C has a large dissociation constant (KD) for IL-8 at anacidic pH. In one embodiment, the dissociation constant of an antibodyof Disclosure C for IL-8 at an acidic pH is, for example, 3 nM or more,but is not limited thereto. In one embodiment, the dissociation constantof an antibody of Disclosure C for IL-8 at an acidic pH is, for example,10 nM or more, but is not limited thereto. In one embodiment, thedissociation constant of an antibody of Disclosure C for IL-8 at anacidic pH is, for example, 30 nM or more, but is not limited thereto.

In one embodiment, it is preferable that an anti-IL-8 antibody ofDisclosure C has a large dissociation constant (KD) for IL-8 at pH 5.8.In one embodiment, the dissociation constant of an antibody ofDisclosure C for IL-8 at pH 5.8 is, for example, 3 nM or more, but isnot limited thereto. In one embodiment, the dissociation constant of anantibody of Disclosure C for IL-8 at pH 5.8 is, for example, 10 nM ormore, but is not limited thereto. In one embodiment, the dissociationconstant of an antibody of Disclosure C for IL-8 at pH 5.8 is, forexample, 30 nM or more, but is not limited thereto.

In one embodiment, it is preferable that the binding affinity of ananti-IL-8 antibody of Disclosure C for IL-8 is greater at a neutral pHthan at an acidic pH.

In one embodiment, the dissociation constant ratio between acidic pH andneutral pH, [KD (acidic pH)/KD (neutral pH)], of an anti-IL-8 antibodyof Disclosure C is, for example, 30 or more, but is not limited thereto.In one embodiment, the dissociation constant ratio between acidic pH andneutral pH, [KD (acidic pH)/KD (neutral pH)], of an anti-IL-8 antibodyof Disclosure C is, for example, 100 or more, for example, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000,4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500, butis not limited thereto.

In one embodiment, the dissociation constant ratio between pH 5.8 and pH7.4, [KD (pH 5.8)/KD (pH 7.4)], of an anti-IL-8 antibody of Disclosure Cis 30 or more, but is not limited thereto. In one embodiment, thedissociation constant ratio between pH 5.8 and pH 7.4, [KD (pH 5.8)/KD(pH 7.4)], of an antibody of Disclosure C is, for example, 100 or more,for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,8500, 9000, or 9500, but is not limited thereto.

In one embodiment, it is preferable that an anti-IL-8 antibody ofDisclosure C has a large dissociation rate constant (koff) at an acidicpH. In one embodiment, the dissociation rate constant of an antibody ofDisclosure C at an acidic pH is, for example, 0.003 (1/s) or more, butis not limited thereto. In one embodiment, the dissociation rateconstant of an antibody of Disclosure C at an acidic pH is, for example,0.005 (1/s) or more, but is not limited thereto. In one embodiment, thedissociation rate constant of an antibody of Disclosure C at an acidicpH is, for example, 0.01 (1/s) or more, but is not limited thereto.

In one embodiment, it is preferable that an anti-IL-8 antibody ofDisclosure C has a large dissociation rate constant (koff) at pH 5.8. Inone embodiment, the dissociation rate constant of an antibody ofDisclosure C at pH 5.8 is, for example, 0.003 (1/s) or more, but is notlimited thereto. In one embodiment, the dissociation rate constant of anantibody of Disclosure C at pH 5.8 is, for example, 0.005 (1/s) or more,but is not limited thereto. In one embodiment, the dissociation rateconstant of an antibody of Disclosure C at pH 5.8 is, for example, 0.01(1/s) or more, but is not limited thereto.

In one embodiment, it is preferable that the anti-IL-8 antibody ofDisclosure C maintains the IL-8-neutralizing activity stably in asolution (for example, in PBS). Whether the activity is maintainedstably in a solution can be assessed by measuring whether theIL-8-neutralizing activity of the antibody of Disclosure C added to thesolution changes before and after storage for a certain period of timeat a certain temperature. In one embodiment, the storage period is, forexample, one, two, three, or four weeks, but is not limited thereto. Inone embodiment, the storage temperature is, for example, 25° C., 30° C.,35° C., 40° C., or 50° C., but is not limited thereto. In oneembodiment, the storage temperature is, for example, 40° C., but is notlimited thereto; and the storage period is, for example, two weeks, butis not limited thereto. In one embodiment, the storage temperature is,for example, 50° C., but is not limited thereto; and the storage periodis, for example, one week, but is not limited thereto.

In one embodiment, it is preferable that the anti-IL-8 antibody ofDisclosure C maintains the IL-8-neutralizing activity stably in vivo(for example, in plasma). Whether the activity is maintained stably invivo can be assessed by measuring whether the IL-8-neutralizing activityof the antibody of Disclosure C added to plasma of an animal (forexample, mouse) or human changes before and after storage for a certainperiod of time at a certain temperature. In one embodiment, the storageperiod is, for example, one, two, three, or four weeks, but is notlimited thereto. In one embodiment, the storage temperature is, forexample, 25° C., 30° C., 35° C., or 40° C., but is not limited thereto.In one embodiment, the storage temperature is, for example, 40° C., butis not limited thereto; and the storage period is, for example, twoweeks, but is not limited thereto.

In one embodiment, the rate of cellular uptake of an anti-IL-8 antibodyof Disclosure C is greater when the antibody forms a complex with IL-8than the antibody alone. The IL-8 antibody of Disclosure C is moreeasily taken up into cells when it is complexed with IL-8 outside ofcells (for example, in plasma) than when not complexed with IL-8.

In one embodiment, it is preferable that the predicted immunogenicity ofan anti-IL-8 antibody of Disclosure C, which is predicted in humanhosts, is reduced. “Low immunogenicity” may mean, without being limitedthereto, for example, that the administered anti-IL-8 antibody does notinduce immune response of a living body in at least half or more of theindividuals administered with a sufficient amount of the antibody for asufficient period of time to achieve therapeutic efficacy. The inductionof immune response may include production of anti-drug antibodies. “Lowanti-drug antibody production” is interchangeable with “lowimmunogenicity”. The immunogenicity level in human can be estimated witha T cell epitope prediction program. Such T cell epitope predictionprograms include Epibase (Lonza), iTope/TCED (Antitope), EpiMatrix(EpiVax), and so on. EpiMatrix is a system for predicting theimmunogenicity of a protein of interest where sequences of peptidefragments are automatically designed by partitioning the amino acidsequence of a protein being analyzed for its immunogenicity into nineamino acids each to predict their ability to bind to eight major MHCClass II alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801,DRB1*1101, DRB1*1301, and DRB1*1501) (De Groot et al., Clin. Immunol.131(2):189-201 (2009)). Sequences in which amino acids of the amino acidsequence of an anti-IL-8 antibody have been modified can be analyzedusing the above-described T cell epitope prediction programs to designsequences with reduced immunogenicity. Preferred sites of amino acidmodification to reduce the immunogenicity of the anti-IL-8 antibody ofDisclosure C include, but are not limited to, the amino acids atposition 81 and/or position 82b according to Kabat numbering in theheavy-chain sequence of the anti-IL-8 antibody shown in SEQ ID NO:78.

In one embodiment, Disclosure C provides methods for enhancingelimination of IL-8 from an individual as compared to when using areference antibody, comprising administering an anti-IL-8 antibody ofDisclosure C to the individual. In one embodiment, Disclosure C relatesto the use of an anti-IL-8 antibody of Disclosure C in the enhancementof the elimination of IL-8 from an individual as compared to when usinga reference antibody. In one embodiment, Disclosure C relates to ananti-IL-8 antibody of Disclosure C for use in the enhancement of theelimination of IL-8 from an individual as compared to when using areference antibody. In one embodiment, Disclosure C relates to the useof an anti-IL-8 antibody of Disclosure C in the production ofpharmaceutical compositions for enhancing the elimination of IL-8 invivo as compared to when using a control antibody. In one embodiment,Disclosure C relates to pharmaceutical compositions comprising ananti-IL-8 antibody of Disclosure C for enhancing the elimination of IL-8as compared to when using a reference antibody. In one embodiment,Disclosure C relates to methods for enhancing the elimination of IL-8 ascompared to when using a reference antibody, comprising administering ananti-IL-8 antibody of Disclosure C to a subject. In the embodiments ofDisclosure C, the reference antibody refers to an anti-IL-8 antibodybefore modification to obtain the antibody of Disclosure C, or anantibody whose IL-8 binding affinity is strong at both acidic andneutral pHs. The reference antibody may be an antibody comprising theamino acid sequence of SEQ ID NOs:83 and 84, or SEQ ID NOs:89 and 87.

In one embodiment, Disclosure C provides pharmaceutical compositionscomprising an anti-IL-8 antibody of Disclosure C, characterized that theanti-IL-8 antibody of Disclosure C binds to IL-8 and then toextracellular matrix. In one embodiment, Disclosure C relates to the useof an anti-IL-8 antibody of Disclosure C in producing pharmaceuticalcompositions characterized that the anti-IL-8 antibody of Disclosure Cbinds to IL-8 and then to extracellular matrix.

In any of the embodiments described above, the anti-IL-8 antibody may bea humanized antibody.

In one aspect, the antibody of Disclosure C comprises the heavy chainvariable region of any one of the embodiments described above and thelight chain variable region of any one of the embodiments describedabove. In one embodiment, the antibody of Disclosure C comprises each ofthe heavy-chain variable region of SEQ ID NO:78 and the light-chainvariable region of SEQ ID NO:79, and also may comprisepost-translational modifications in their sequences.

In a further aspect, an anti-IL-8 antibody according to any one of theembodiments described above may incorporate, singly or in combination,any of the features described in Sections 1 to 7 below.

1. Chimeric Antibody and Humanized Antibody

In certain embodiments, an antibody provided in Disclosure C may be achimeric antibody. Certain chimeric antibodies are described, forexample, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.Acad. Sci. USA 81:6851-6855 (1984). In one example, a chimeric antibodymay comprise a non-human variable region (e.g., a variable regionderived from a mouse, a rat, a hamster, a rabbit, or a non-human primatesuch as a monkey) and a human constant region.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity inhumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable regions in which HVRs, e.g., CDRs (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody may besubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), for example, toretain or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro et al., Front. Biosci. 13:1619-1633 (2008), and arefurther described, for example, in Riechmann et al., Nature 332:323-329(1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiriet al., Methods 36:25-34 (2005) (describing specificity determiningregion (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991)(describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005)(describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005)and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the“guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to framework regions selected using the “best-fit”method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal., J. Immunol., 151:2623 (1993)); and framework regions derived fromscreening FR libraries (see, e.g., Baca et al., J. Biol. Chem.272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618(1996)).

2. Antibody Fragments

In certain embodiments, an antibody provided in Disclosure C may be anantibody fragment. Antibody fragments include, but are not limited to,Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragmentsdescribed below. For a review of certain antibody fragments, see Hudsonet al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, seefor example, Pluckthün, in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody may be a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g., E. coli or phage), as described within thescope of the description of Disclosure C herein.

3. Human Antibody

In certain embodiments, an antibody provided in Disclosure C may be ahuman antibody. Human antibodies can be prepared by various techniquesknown in the art. Human antibodies are described in general terms in vanDijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) andLonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies may beprepared by administering an immunogen to a transgenic animal that hasbeen modified to produce intact human antibodies or intact antibodieswith human variable regions in response to antigenic challenge. Suchanimals typically contain all or a portion of the humans immunoglobulinloci, which replace the immunoglobulin loci of the animal (non-human),or are present extrachromosomally or integrated randomly into theanimal's chromosomes. In such transgenic mice, the immunoglobulin lociof the animal (non-human) have generally been inactivated. For review ofmethods for obtaining human antibodies from transgenic animals, seeLonberg, Nat. Biotech. 23:1117-1125 (2005). See also U.S. Pat. Nos.6,075,181 and 6,150,584 for XENOMOUSE™ technology; U.S. Pat. No.5,770,429 for HUMAB™ technology; U.S. Pat. No. 7,041,870 for K-M MOUSE™technology, and US Patent Appl. Publ. No. US 2007/0061900 forVELOCIMOUSE™ technology.

Human variable regions from intact antibodies produced by such animalsmay be further modified, for example, by combining with a differenthuman constant region. Human antibodies can also be prepared byhybridoma-based methods. Human myeloma and mouse-human heteromyelomacell lines for the production of human monoclonal antibodies aredescribed, for example, in Kozbor, J. Immunol. 133:3001 (1984); Brodeuret al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol. 147:86 (1991). Human antibodies generated via human B-cellhybridoma are described in Li et al., Proc. Natl. Acad. Sci. USA103:3557-3562 (2006). Additional methods include, for example, themethod described in U.S. Pat. No. 7,189,826, for the production ofmonoclonal human IgM antibodies from hybridoma cell lines, as well as,for example, the method described in Ni, Xiandai Mianyixue, 26(4):265-268 (2006), for human-human hybridomas. Human hybridomatechnology (trioma technology) is also described in Vollmers et al.,Histol. and Histopath. 20(3):927-937 (2005) and Vollmers et al., Methodsand Findings in Experimental and Clinical Pharmacology 27(3):185-91(2005). Human antibodies may also be generated by isolating Fv clonevariable domain sequences selected from human-derived phage displaylibraries. Such variable domain sequences can be combined with a desiredhuman constant domain. Techniques for selecting human antibodies fromantibody libraries are described below.

4. Library-Derived Antibodies

Antibodies of Disclosure C can be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, various methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingdesired binding characteristics. Such methods are reviewed in Hoogenboomet al., in Meth. Mol. Biol. 178:1-37 (O'Brien et al., ed., Human Press,Totowa, N. J., 2001), as wells as, for example, in McCafferty et al.,Nature 348:552-554 (1990); Clackson et al., Nature 352:624-628 (1991);Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, inMeth. Mol. Biol. 248:161-175 (Lo, ed., Human Press, Totowa, N. J.,2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J.Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA101(34):12467-12472 (2004); and Lee et al., J. Immunol. Meth.284(1-2):119-132 (2004).

In certain phage display methods, repertoires of VH and VL codingsequences may be separately cloned by polymerase chain reaction (PCR)and recombined randomly in phage libraries. The resulting phagelibraries are screened for antigen-binding phage as described in Winteret al., Ann. Rev. Immunol. 12:433-455 (1994). Phage typically displayantibody fragments, either as single-chain Fv (scFv) fragments or as Fabfragments.

Alternatively, the naive repertoire can be cloned (for example, fromhuman) to provide a single source of antibodies to a wide range ofnon-self and also self antigens without any immunization as described byGriffiths et al., EMBO J. 12:725-734 (1993).

Finally, naïve libraries can also be constructed synthetically bycloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequences to encode the highly variable CDR3regions and to accomplish rearrangement in vitro (see below andHoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992); patentpublications that describe human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373; and US Appl. Publ. Nos. 2005/0079574,2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764,2007/0292936, and 2009/0002360. Here, antibodies or antibody fragmentsisolated from human antibody libraries are considered to be humanantibodies or human antibody fragments.

5. Multispecific Antibody

In certain embodiments, an antibody provided according to Disclosure Cmay be, for example, a multispecific antibody such as a bispecificantibody. Multispecific antibodies are monoclonal antibodies that havebinding specificities for at least two different sites.

In certain embodiments, one of the binding specificities is for IL-8,and the others are for any other antigens.

In certain embodiments, bispecific antibodies may bind to two differentepitopes on IL-8. Bispecific antibodies may also be used to localizecytotoxic agents to cells that express IL-8. Bispecific antibodies maybe prepared as full length antibodies or as antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305:537 (1983)); WO 93/08829; and Traunecker et al., EMBOJ. 10:3655 (1991)) and the “knob-in-hole” method (see U.S. Pat. No.5,731,168). Multispecific antibodies can be made by using electrostaticsteering effects to prepare Fc-heterodimeric molecules (WO2009/089004A1), by crosslinking two or more antibodies or fragments(U.S. Pat. No. 4,676,980; and Brennan et al., Science 229:81 (1985)), byusing leucine zippers to produce bispecific antibodies (Kostelny et al.,J. Immunol. 148(5):1547-1553 (1992)), by using “diabody” technology tomake bispecific antibody fragments (Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993)), by using single-chain Fv (sFv) dimers(Gruber et al., J. Immunol. 152:5368 (1994)), or other methods.Preparation of trispecific antibodies is described, for example, in Tuttet al., J. Immunol. 147:60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies”, are also included herein (see,e.g., US 2006/0025576).

Within the scope of the description of Disclosure C herein, the antibodyor antibody fragment also includes a “Dual Acting FAb” or “DAF”comprising an antigen binding site that binds to IL-8 as well asanother, different antigen (see US 2008/0069820, for example).

6. Antibody Variants

Amino acid sequence variants of an antibody can be prepared byintroducing appropriate modifications into a nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions, and/or insertions, and/or substitutionsof residues in the amino acid sequence of the antibody. A finalconstruct can be attained with any combination of deletion, insertion,and substitution, as long as the final construct is an antibody that hasthe desired properties described in the context of Disclosure C.

In one embodiment, Disclosure C provides antibody variants having one ormore amino acid substitutions. Such substitution sites may be anypositions in an antibody. Amino acids for conservative substitutions areshown in Table 10 under the heading of “conservative substitutions”.Amino acids for typical substitutions that result in more substantialchanges are shown in Table 10 under the heading of “typicalsubstitutions”, and as further described in reference of amino acid sidechain classes.

TABLE 10 Original Typical Conservative Residue Substitution SubstitutionAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln: Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutralhydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic:His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro;and (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Amino acid insertions include fusion of a polypeptide comprising one,two, or three to one hundred or more residues at the N terminus and/or Cterminus, as well as insertion of one or more amino acid residues into asequence. Antibodies with such terminal insertion include, for example,antibodies with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include those that result from N- orC-terminal fusion of the antibody to an enzyme (for example, ADEPT) or apolypeptide that increases plasma half-life of antibody.

7. Glycosylated Variants

In one embodiment, antibodies provided according to Disclosure C may beglycosylated antibodies. Glycosylation sites can be added to or deletedfrom an antibody by altering amino acid sequences in such a way as tocreate or remove glycosylation sites.

When an antibody comprises an Fc region, the sugar chain attachedthereto can be altered. Naïve antibodies produced by mammalian cellstypically contain a branched, biantennary oligosaccharide, which isattached by an N-linkage to Asn297 of the CH2 domain of the Fc region(see Wright et al. TIBTECH 15:26-32 (1997)). The oligosaccharideincludes, for example, mannose, N-acetylglucosamine (GlcNAc), galactose,and sialic acid, as well as fucose attached to GlcNAc in the “stem” ofthe biantennary oligosaccharide structure. In one embodiment, theoligosaccharide in the antibody of Disclosure C is modified to createantibody variants having certain improved properties.

8. Fc Region Variants

In one embodiment, one or more amino acid modifications are introducedinto the Fc region of an antibody provided according to Disclosure C,thereby generating an Fc region variant. Fc region variants includethose that have a modification (for example, a substitution) of one,two, three, or more amino acids in a native human Fc region sequence(for example, the Fc region of human IgG1, IgG2, IgG3, or IgG4).

An anti-IL-8 antibody of Disclosure C may contain an Fc region having atleast one of the following five properties, without being limitedthereto: (a) increased binding affinity for FcRn of the Fc regionrelative to the binding affinity for FcRn of a native Fc region atacidic pH; (b) reduced binding affinity of the Fc region forpre-existing ADA relative to the binding affinity of a native Fc regionfor the pre-existing ADA; (c) increased plasma half-life of the Fcregion relative to the plasma half-life of a native Fc region; (d)reduced plasma clearance of the Fc region relative to the plasmaclearance of a native Fc region; and (e) reduced binding affinity of theFc region for an effector receptor relative to the binding affinity of anative Fc region for the effector receptor. In some embodiments, the Fcregion has 2, 3 or 4 of the above-listed properties. In one embodiment,Fc region variants include those having an increased FcRn-bindingaffinity at an acidic pH. Fc region variants with increased FcRn-bindingaffinity include, but are not limited to, Fc region variants whoseFcRn-binding affinity is increased up to 2-fold, 3-fold, 4-fold, 5-fold,10-fold, 15-fold, 20-fold, 30-fold, 50-fold, or 100-fold as compared tothe FcRn-binding affinity of an antibody comprising the native IgG Fcregion.

In one embodiment, an Fc region variant includes a safe and advantageousFc region variant that does not bind to pre-existing ADA, and at thesame time has improved plasma retention. As used in the context ofDisclosure C, the term “ADA” refers to an endogenous antibody havingbinding affinity for an epitope on a therapeutic antibody. As used inthe context of Disclosure C, the term “pre-existing ADA” refers to adetectable anti-drug antibody present in a patient's blood prior toadministration of a therapeutic antibody to the patient. Pre-existingADA includes the rheumatoid factor. Fc region variants with low bindingaffinity for pre-existing ADA include, but are not limited to, Fc regionvariants whose ADA-binding affinity is reduced to 1/10 or less, 1/50 orless, or 1/100 or less as compared to the ADA-binding affinity of anantibody comprising the native IgG Fc region.

In one embodiment, an Fc region variant includes an Fc region variantswhose binding affinity for complement proteins is low or that do notbind to complement proteins. Complement proteins include C1q. Fc regionvariants with low binding affinity for complement proteins include, butare not limited to, Fc region variants whose binding affinity forcomplement proteins is reduced to 1/10 or less, 1/50 or less, or 1/100or less as compared to the complement protein-binding affinity of anantibody comprising a native IgG Fc region.

In one embodiment, an Fc region variant includes an Fc region variantwhose binding affinity for effector receptors is low or that does nothave the binding affinity for an effector receptor. The effectorreceptors include, but are not limited to, FcγRI, and FcγRIII. FcγRIincludes, but is not limited to, FcγRIa, FcγRIb, and FcγRIc, as well assubtypes thereof. FcγRII includes, but is not limited to, FcγRIIa (whichhas two allotypes: R131 and H131) and FcγRIIb. FcγRIII includes, but isnot limited to, FcγRIIIa (which has two allotypes: V158 and F158) andFcγRIIIb (which has two allotypes: FcγRIIIb-NA1 and FcγRIIIb-NA2). Fcregion variants with low binding affinity for effector receptorsinclude, but are not limited to, Fc region variants whose bindingaffinity for effector receptors is reduced to at least 1/10 or less,1/50 or less, or 1/100 or less as compared to the binding affinity of anantibody comprising a native IgG Fc region.

In one embodiment, an Fc region variant includes an Fc region comprisingone or more amino acid substitutions at any of the positions of thegroup consisting of positions 235, 236, 239, 327, 330, 331, 428, 434,436, 438, and 440, according to EU numbering as compared to the nativeFc region.

In one embodiment, an Fc region variant includes an Fc region comprisingamino acid substitutions at positions 235, 236, 239, 428, 434, 436, 438,and 440, according to EU numbering as compared to the native Fc region.

In one embodiment, an Fc region variant includes an Fc region comprisingamino acid substitutions at positions 235, 236, 327, 330, 331, 428, 434,436, 438, and 440, according to EU numbering as compared to the nativeFc region.

In one embodiment, an Fc region variant includes an Fc region comprisingone or more amino acid substitutions selected from the group consistingof: L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T,Q438R, and S440E.

In one embodiment, an Fc region variant includes an Fc region comprisingthe amino acid substitutions of M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, an Fc region variant includes an Fc region comprisingthe amino acid substitutions of L235R, G236R, S239K, M428L, N434A,Y436T, Q438R, and S440E.

In one embodiment, an Fc region variant includes an Fc region comprisingthe amino acid substitutions of L235R, G236R, A327G, A330S, P331S,M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises theamino acid sequence of SEQ ID NO:80 and/or the amino acid sequence ofSEQ ID NO:82. The anti-IL-8 antibody comprising the amino acid sequenceof SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may be ananti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. Theanti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80and/or the amino acid sequence of SEQ ID NO:82 may maintainIL-8-neutralizing activity stably in vivo (for example, in plasma). Theanti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80and/or the amino acid sequence of SEQ ID NO:82 may be an antibody withlow immunogenicity. The anti-IL-8 antibody comprising the amino acidsequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82may contain an Fc region whose FcRn-binding affinity at an acidic pH(e.g., pH 5.8) is increased as compared to the FcRn-binding affinity ofa native Fc region at the acidic pH. The anti-IL-8 antibody comprisingthe amino acid sequence of SEQ ID NO:80 and/or the amino acid sequenceof SEQ ID NO:82 may contain an Fc region whose binding affinity forpre-existing ADA is reduced as compared to the binding affinity of anative Fc region for pre-existing ADA. The anti-IL-8 antibody comprisingthe amino acid sequence of SEQ ID NO:80 and/or the amino acid sequenceof SEQ ID NO:82 may contain an Fc region whose half-life in plasma isprolonged as compared to that of a native Fc region. The anti-IL-8antibody comprising the amino acid sequence of SEQ ID NO:80 and/or theamino acid sequence of SEQ ID NO:82 may contain an Fc region whosebinding affinity for effector receptors is reduced as compared to thatof a native Fc region. In a further embodiment, the anti-IL-8 antibodycomprises a combination of any 2, 3, 4, 5, 6, or all 7 of above-listedproperties.

In one embodiment, an anti-IL-8 antibody of Disclosure C comprises theamino acid sequence of SEQ ID NO:81 and/or the amino acid sequence ofSEQ ID NO:82. The anti-IL-8 antibody comprising the amino acid sequenceof SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may be ananti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. Theanti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81and/or the amino acid sequence of SEQ ID NO:82 may maintainIL-8-neutralizing activity stably in vivo (for example, in plasma). Theanti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81and/or the amino acid sequence of SEQ ID NO:82 may be an antibody withlow immunogenicity. The anti-IL-8 antibody comprising the amino acidsequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82may contain an Fc region whose FcRn-binding affinity at an acidic pH isincreased as compared to the FcRn-binding affinity of a native Fcregion. The anti-IL-8 antibody comprising the amino acid sequence of SEQID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may contain anFc region whose binding affinity for pre-existing ADA is reduced ascompared to the binding affinity of a native Fc region for pre-existingADA. The anti-IL-8 antibody comprising the amino acid sequence of SEQ IDNO:81 and/or the amino acid sequence of SEQ ID NO:82 may contain an Fcregion whose half-life in plasma is prolonged as compared to that of anative Fc region. The anti-IL-8 antibody comprising the amino acidsequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82may contain an Fc region whose binding affinity for effector receptorsis reduced as compared to that of a native Fc region. In a furtherembodiment, the anti-IL-8 antibody comprises a combination of any 2, 3,4, 5, 6, or all 7 of above-listed properties.

In certain embodiments, Disclosure C encompasses an antibody variantthat possesses some but not all effector functions. The antibody variantcan be a desirable candidate for cases in which certain effectorfunctions (such as complement and ADCC) are unnecessary or deleterious.In vitro and/or in vivo cytotoxicity assays known in the art canroutinely be conducted to confirm the reduction/complete loss of CDCand/or ADCC activities. For example, Fc receptor (FcR) binding assayscan be conducted to confirm that an antibody lacks FcγR binding (hencelacking ADCC activity), but retains FcRn binding ability.

The primary cultured cells for mediating ADCC and NK cells expressFcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch et al., Annu. Rev. Immunol. 9:457-492 (1991). Non-limitingexamples of in vitro assays for assessing ADCC activity of a molecule ofinterest are described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom.et al., Proc. Natl Acad. Sci. USA 83:7059-7063 (1986), Hellstrom et al.,Proc. Natl Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337,and Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive isotope assays are available forassessing effector cell function (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.; and CytoTox 96™ non-radioactive cytotoxicityassay (Promega, Madison, Wis.)). Effector cells useful for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells.

Alternatively or additionally, ADCC activity of an antibody variant ofinterest may be assessed in vivo, for example, in an animal model asdisclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656(1998). C1q binding assays may also be carried out to confirm that theantibody is unable to bind C1q and lacks CDC activity. See, e.g., C1qand C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assesscomplement activation, a CDC assay may be performed (see, e.g.,Gazzano-Santoro et al., J. Immunol. Meth. 202:163 (1996); Cragg et al.,Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743(2004)). FcRn binding and in vivo clearance/half life determinations canbe performed using methods known in the art (see, e.g., Petkova et al.,Intl. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector functions include those withsubstitution of one or more of Fc region residues at position 238, 265,269, 270, 297, 327 or 329 (U.S. Pat. No. 6,737,056). Such Fc regionvariants include Fc region variants with substitutions at two or more ofresidues at position 265, 269, 270, 297 or 327, including the so-called“DANA” Fc region variants with substitution of residues 265 and 297 toalanine (U.S. Pat. No. 7,332,581).

Antibody variants with improved or diminished binding to FcR groups aredescribed below. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312,and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

Antibodies with increased blood half lives and improved FcRn binding atan acidic pH are described in US2005/0014934. The described antibodiescomprise an Fc region with one or more substitutions that improvebinding of the Fc region to FcRn. Such Fc region variants include thosewith substitutions at one or more of positions selected from 238, 256,265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376,378, 380, 382, 413, 424 or 434 in an Fc region, for example,substitution of position 434 in an Fc region (U.S. Pat. No. 7,371,826).

See also Duncan et al., Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 for other examplesof Fc region variants.

9. Antibody Derivatives

In certain embodiments, an antibody provided in Disclosure C may befurther modified to contain additional nonproteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the antibody include, but are not limited, to watersoluble polymers. Examples of water soluble polymers include, but arenot limited to, polyethylene glycol (PEG), copolymers of ethylene glycolor propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyamino acids (eitherhomopolymers or random copolymers), poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

Polyethylene glycol propionaldehyde has advantages in industrializationdue to its stability in water. This polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymers areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivertization can bedetermined based on considerations including, but not limited to, theparticular properties or functions of the antibody to be improved, whenthe antibody derivative is used in a defined therapy, etc.

In another embodiment, conjugates of an anti-IL-8 antibody of DisclosureC and nonproteinaceous moiety that may be selectively heated by exposureto radiation may be provided. In one embodiment, the nonproteinaceousmoiety is, for example, a carbon nanotube (see, e.g., Kam et al., Proc.Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be ofany wavelength and includes, without being limited thereto, wavelengthsthat are harmless to humans but can heat the nonproteinaceous moiety toa temperature so as to kill cells proximal to theantibody-nonproteinaceous moiety.

B. Recombination Methods and Compositions

Anti-IL-8 antibodies of Disclosure C may be produced using recombinantmethods and compositions, for example, as described in U.S. Pat. No.4,816,567. One embodiment provides isolated nucleic acid(s) encoding ananti-IL-8 antibody which are presented as Disclosure C. Such nucleicacid(s) may encode an amino acid sequence comprising the VL of theantibody and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In one embodiment, a host cellcomprising such nucleic acid(s) is provided. In one such embodiment, ahost cell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes the VL of the antibody and the VHof an antibody, or (2) a first vector comprising a nucleic acid thatencodes the VL of an antibody and a second vector comprising a nucleicacid that encodes the VH of the antibody.

In one embodiment, the host is eukaryotic (e.g., a Chinese Hamster Ovary(CHO) cell or lymphoid cell (e.g., Y0, NS0, SP20 cell)).

In one embodiment, a method of producing an anti-IL-8 antibody ofDisclosure C is provided, wherein the method comprises culturing a hostcell comprising a nucleic acid encoding the anti-IL-8 antibody asprovided above, under conditions suitable for expressing the antibody,and optionally recovering the antibody (e.g., from the host cell or hostcell culture medium).

For recombinant production of an anti-IL-8 antibody, nucleic acid(s)encoding an antibody, for example, as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such nucleic acid(s) may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that specifically bind to nucleic acids encoding the heavy andlight chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described within thescope of the description of Disclosure C herein. For example, antibodiesmay be produced in bacteria, in particular, when glycosylation and Fceffector function are not needed. For expression of antibody fragmentsand polypeptides in bacteria, see for example, U.S. Pat. Nos. 5,648,237,5,789,199, and 5,840,523 (See also Charlton, Methods in MolecularBiology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N. J., 2003),pp. 245-254, for expression of antibody fragments in E. coli). Afterexpression, the antibody may be isolated from the bacterial cell pastein a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for cloning or expression ofantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized”, which enable production ofantibodies with a partially or fully human glycosylation pattern. SeeGerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat.Biotech. 24:210-215 (2006).

Suitable host cells for the expression of a glycosylated antibody arealso derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Without particular limitations, baculovirus is used inconjunction with insect cells for transfection of Spodoptera frugiperdacells and numerous baculoviral strains have been identified.

Plant cell cultures can also be utilized as hosts. See U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension are useful. Other examplesof useful mammalian host cells are monkey kidney CV1 line transformed bySV40 (COS-7); human embryonic kidney line (293 cells as described inGraham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells(BHK); mouse sertoli cells (TM4 cells as described in Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African greenmonkey kidney cells (VERO-76); human cervical carcinoma cells (HELA);canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lungcells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT060562); TM cells, as described in Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982); MRCS cells; and FS4 cells.

Other useful mammalian host cell lines include Chinese hamster ovary(CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 andSp20, but are not limited thereto. For a review of other mammalian hostcell lines suitable for antibody production, see Yazaki and Wu, Methodsin Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

An antibody of Disclosure C produced by culturing such host cells asdescribed above to carry nucleic acids that encode the antibody underconditions that are suitable for antibody expression may be isolatedfrom inside or outside of the host cells (media, milk, etc.), andpurified as a substantially pure homogeneous antibody.Isolation/purification methods that are generally used to purifypolypeptides can be appropriately used to isolate and purify theantibody; however, the methods are not limited to the above example. Theantibody can be appropriately separated and purified, for example, byappropriately selecting and combining column chromatography, filters,ultrafiltration, salting out, solvent precipitation, solvent extraction,distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectric focusing, dialysis, and recrystallization,without being limited thereto. Chromatography includes, but is notlimited to, affinity chromatography, ion exchange chromatography,hydrophobic chromatography, gel filtration chromatography, reverse phasechromatography, and adsorption chromatography. Such chromatography canbe performed using liquid chromatography, for example, HPLC and FPLC.Columns for use in affinity chromatography include, but are not limitedto, Protein A column and Protein G column. Protein A columns include,but are not limited to, Hyper D, POROS, Sepharose F. F. (Pharmacia) andso on.

Focusing on the characteristics of anti-IL-8 antibodies such asincreased extracellular matrix-binding activity and enhanced cellularuptake of the complex described above, Disclosure C provides methods forselecting antibodies with increased extracellular matrix-binding andantibodies with enhanced cellular uptake. In one embodiment, DisclosureC provides methods for producing an anti-IL-8 antibody comprisingvariable region whose binding activity to IL-8 is in an pH-dependentmanner, which comprise the steps of: (a) assessing the binding betweenanti-IL-8 antibody and extracellular matrix; (b) selecting an anti-IL-8antibody that strongly binds to extracellular matrix; (c) culturing ahost that comprises a vector carrying a nucleic acid encoding theantibody; and (d) isolating the antibody from the culture medium.

Binding with extracellular matrix can be assessed by any methods withoutparticular limitations, as long as they are known to those of ordinaryskill in the art. For example, assays can be carried out using an ELISAsystem for detecting the binding between an antibody and extracellularmatrix, where the antibody is added to an extracellularmatrix-immobilized plate and a labeled antibody against the antibody isadded thereto. Alternatively, such assays can be performed, for example,using an electrochemiluminescence (ECL) method in which a mixture of theantibody and a ruthenium antibody is added to an extracellularmatrix-immobilized plate and the binding between the antibody andextracellular matrix is assessed based on the electrochemiluminescenceof ruthenium.

The anti-IL-8 antibody being assessed for extracellular matrix bindingin step (i) above may be the antibody by itself or in contact with IL-8.“Selecting an anti-IL-8 antibody that strongly binds to extracellularmatrix” in step (ii) means that an anti-IL-8 antibody is selected basedon the criterion that a value representing the binding betweenextracellular matrix and the anti-IL-8 antibody is higher than a valuerepresenting the binding between extracellular matrix and the controlantibody in the assessment of extracellular matrix binding, and may be,for example, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more; however, the ratiois not particularly limited to the examples above. Other than thepresence of IL-8, the conditions are preferably the same in the step ofassessing the binding between an anti-IL-8 antibody and extracellularmatrix. The control anti-IL-8 antibody for use in comparing severalmodified anti-IL-8 antibodies may be the unmodified anti-IL-8 antibody.In this case, the conditions are preferably the same other than thepresence of IL-8. Specifically, in one embodiment, Disclosure C includesselecting an antibody with a higher value representing extracellularmatrix binding from several anti-IL-8 antibodies that are not in contactwith IL-8. In another embodiment, Disclosure C includes selecting anantibody with a higher value representing extracellular matrix bindingfrom several anti-IL-8 antibodies that are in contact with IL-8. In analternative embodiment, “selecting an anti-IL-8 antibody that stronglybinds to extracellular matrix” in step (ii) means that an antibody maybe selected based on the criterion that the binding between an antibodyand extracellular matrix varies depending on the presence of IL-8, whenassessing extracellular matrix binding. The ratio of a valuerepresenting the extracellular matrix binding of an anti-IL-8 antibodyin contact with IL-8 to a value representing the extracellular matrixbinding of an anti-IL-8 antibody not in contact with IL-8 may be, forexample, 2 to 1000. Furthermore, the ratio between the values may be 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800,900, or 1000.

C. Assays

Anti-IL-8 antibodies provided within the scope of Disclosure C describedherein can be identified, screened, or characterized in terms of theirphysical/chemical properties and/or biological activities by variousmethods known in the art.

1. Binding Assays and Other Assays

In one aspect, the antibodies of Disclosure C can be assessed for theirantigen-binding activity by known methods, for example, ELISA, Westernblotting, kinetic exclusion assay (KinExA™), and surface plasmonresonance using a device such as BIACORE (GE Healthcare).

In one embodiment, the binding affinity can be measured using BIACORET200 (GE Healthcare) in the following manner. An appropriate amount of atrapping protein (for example, Protein A/G (PIERCE)) is immobilized ontoa sensor chip CM4 (GE Healthcare) by the amine-coupling method, and anantibody of interest is allowed to be captured. Then, a diluted antigensolution and running buffer (as a reference solution: for example, 0.05%tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4) are injected to interactantigen molecules with the antibody trapped on the sensor chip. Thesensor chip is regenerated using 10 mM glycine HCl solution (pH 1.5).Measurements are performed at a pre-determined temperature (for example,37° C., 25° C., or 20° C.). The association rate constant kon (1/Ms) anddissociation rate constant koff (1/s), both of which are kineticparameters, are calculated from sensorgrams obtained by measurement. TheKD (M) of each antibody for the antigen is calculated based on theseconstants. Each parameter is calculated using the BIACORE T200Evaluation Software (GE Healthcare).

In one embodiment, IL-8 can be quantitated as described below. Ananti-human IL-8 antibody comprising the mouse IgG constant region isimmobilized onto a plate. A solution comprising IL-8 bound to ahumanized anti-IL-8 antibody, which does not compete with theabove-described anti-human IL-8 antibody, is aliquoted to theimmobilized plate. After stirring, a biotinylated anti-human Ig κ klight chain antibody is added and allowed to react for a certain periodof time. Then, SULFO-Tag-labeled streptavidin is further added andallowed to react for a certain period of time. Then, assay buffer isadded and immediately measurement is performed with SECTOR Imager 2400(Meso Scale Discovery).

2. Activity Assays

In one aspect, assays are provided to identify an anti-IL-8 antibodyhaving a biological activity. The biological activity includes, forexample, IL-8-neutralizing activity and the activity of blocking IL-8signals. The Disclosure C also provides antibodies with such biologicalactivity in vivo and/or ex vivo.

In one embodiment, the method for determining the level of IL-8neutralization is not particularly limited and it can also be determinedby the methods described below. PathHunter™ CHO-K1 CXCR2 β-Arrestin CellLine (DiscoveRx, Cat. #93-0202C2) is an artificial cell line created toexpress human CXCR2 known as a human IL-8 receptor and emitchemiluminescence when receiving signals by human IL-8. When human IL-8is added to a culture medium of the cells, chemiluminescence is emittedfrom the cells in a manner that depends on the concentration of addedhuman IL-8. When human IL-8 is added in combination with an anti-humanIL-8 antibody to the culture medium, the chemiluminescence of the cellsis reduced or undetectable as compared to when the antibody is notadded, since the anti-human IL-8 antibody can block the IL-8 signaltransduction. Specifically, the stronger the human IL-8-neutralizingactivity of the antibody is, the weaker the level of chemiluminescenceis; and the weaker the human IL-8-neutralizing activity of the antibodyis, the greater the level of chemiluminescence is. Thus, the humanIL-8-neutralizing activity of the anti-human IL-8 antibody can beassessed by examining the difference described above.

D. Pharmaceutical Formulations

Pharmaceutical formulations comprising an anti-IL-8 antibody asdescribed within the scope of the description Disclosure C herein may beprepared by mixing an anti-IL-8 antibody having the desired degree ofpurity with one or more optional pharmaceutically acceptable carriers(see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)) in the form of lyophilized formulations or aqueous solutionformulations.

Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and includewithout being limited to buffers such as phosphate, citrate, histidine,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride; benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular-weight (less than about 10 residues)polypeptides; proteins such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™, orpolyethylene glycol (PEG).

Exemplary pharmaceutically acceptable carriers herein further includeinterstitial drug dispersion agents such as soluble hyaluronidaseglycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidaseglycoproteins, such as rHuPH20 (HYLENEX™, Baxter International, Inc.).Certain exemplary sHASEGPs and methods of use, including rHuPH20, aredescribed in US Appl. Publ. Nos. 2005/0260186 and 2006/0104968. In oneaspect, a sHASEGP is combined with one or more glycosaminoglycanasessuch as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908; and the WO2006/044908formulations include a histidine-acetate buffer.

The formulation within the scope of Disclosure C herein may also containmore than one active ingredients as necessary for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. Such active ingredients aresuitably present in combination in amounts that are effective for thepurpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the anti-IL8 antibody of the DisclosureC, in which the matrices are in the form of shaped articles, forexample, films or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, for example, byfiltration through sterile filtration membranes.

E. Therapeutic Methods and Compositions

In some embodiments, the anti-IL-8 antibodies provided according toDisclosure C are used in therapeutic methods.

In one aspect, an anti-IL-8 antibody for use as a pharmaceuticalcomposition is provided. In an alternative aspect, an anti-IL-8 antibodyfor use in treating a disease where IL-8 is present in an excessiveamount is provided. In one embodiment, an anti-IL-8 antibody for use inmethods for treating a disease where IL-8 is present in an excessiveamount is provided. In one embodiment, Disclosure C provides methods fortreating an individual with a disease where IL-8 is present in anexcessive amount (for example, a disease caused by the presence ofexcessive IL-8), which comprises administering an effective amount of ananti-IL-8 antibody to the individual. In another embodiment, DisclosureC provides anti-IL-8 antibodies for use in such methods. In oneembodiment, Disclosure C relates to a pharmaceutical compositioncomprising an effective amount of an anti-IL-8 antibody, which is usedto treat a disease where IL-8 is present in an excessive amount. In oneembodiment, Disclosure C relates to the use of an anti-IL-8 antibody inproducing a pharmaceutical composition for a disease where IL-8 ispresent in an excessive amount. In one embodiment, Disclosure C relatesthe use of an effective amount of an anti-IL-8 antibody in treating adisease where IL-8 is present in an excessive amount. Diseases whereIL-8 is present in an excessive amount include, but are not limited to,inflammatory skin diseases such as inflammatory keratosis (psoriasis,etc.), atopic dermatitis, and contact dermatitis; autoimmune diseasessuch as chronic inflammatory diseases including chronic rheumatoidarthritis, systemic lupus erythematosus (SLE), and Behcet disease;inflammatory bowel diseases such as Crohn's disease and ulcerativecolitis; inflammatory liver diseases such as hepatitis B, hepatitis C,alcoholic hepatitis, and allergic hepatitis induced by drugs;inflammatory kidney diseases such as glomerular nephritis; inflammatoryrespiratory diseases such as bronchitis and asthma; chronic inflammatoryvascular disease such as atherosclerosis; multiple sclerosis; oralulcer; chorditis; inflammation induced by an artificial organ/artificialblood vessel; a malignant tumor such as ovarian cancer, lung cancer,prostate cancer, stomach cancer, breast cancer, melanoma, head and neckcancer, and kidney cancer; sepsis caused by infection; cystic fibrosis;pulmonary fibrosis; and acute lung injury.

In an alternative embodiment, Disclosure C provides an anti-IL-8antibody for use in suppressing the accumulation of IL-8 with biologicalactivity. “Suppressing the accumulation of IL-8” may be achieved bypreventing the amount of pre-existing IL-8 in vivo from increasing or byreducing the amount of pre-existing IL-8 in vivo. In one embodiment, theDisclosure C provides an anti-IL-8 antibody for suppressing theaccumulation of IL-8 in an individual to suppress the accumulation ofIL-8 with biological activity. Here, “IL-8 present in vivo” may refer toIL-8 complexed with anti-IL-8 antibody or free IL-8; alternatively, itmay refer to total IL-8 as its sum. Herein, “present in vivo” may mean“secreted to the outside of the cells in vivo.” In one embodiment,Disclosure C provides a method for suppressing the accumulation of IL-8with biological activity, which comprises the step of administering aneffective amount of an anti-IL-8 antibody. In one embodiment, DisclosureC relates to a pharmaceutical composition for suppressing theaccumulation of IL-8 with biological activity, which comprises aneffective amount of an anti-IL-8 antibody. In one embodiment, DisclosureC relates to the use of an anti-IL-8 antibody in producing apharmaceutical composition for suppressing the accumulation of IL-8 withbiological activity. In one embodiment, Disclosure C relates to the useof an effective amount of an anti-IL-8 antibody in suppressing theaccumulation of IL-8 with biological activity. In one embodiment, ananti-IL-8 antibody of Disclosure C suppresses the accumulation of IL-8as compared to an anti-IL-8 antibody that does not have pH-dependentbinding activity. In the above-described embodiments, the “individual”is preferably a human.

In an alternative embodiment, Disclosure C provides an anti-IL-8antibody for use in inhibiting angiogenesis (e.g., neoangiogenesis). Inone embodiment, Disclosure C provides a method for inhibitingneoangiogenesis in an individual which comprises administering aneffective amount of an anti-IL-8 antibody to the individual, and alsoprovides an anti-IL-8 antibody for use in the method. In one embodiment,Disclosure C relates to a pharmaceutical composition for inhibitingneoangiogenesis which comprises an effective amount of an anti-IL-8antibody. In one embodiment, Disclosure C relates to the use of ananti-IL-8 antibody in producing a pharmaceutical composition forinhibiting neoangiogenesis. In one embodiment, Disclosure C relates tothe use of an effective amount of an anti-IL-8 antibody in inhibitingneoangiogenesis. In the above-described embodiments, the “individual” ispreferably a human.

In an alternative aspect, Disclosure C provides an anti-IL-8 antibodyfor use in inhibiting the facilitation of neutrophil migration. In oneembodiment, Disclosure C provides a methods for inhibiting thefacilitation of neutrophil migration in an individual, which comprisesadministering an effective amount of an anti-IL-8 antibody to theindividual; and also provides an anti-IL-8 antibody for use in themethod. In one embodiment, Disclosure C relates to pharmaceuticalcompositions for inhibiting facilitation of neutrophil migration in anindividual, which comprise an effective amount of an anti-IL-8 antibody.In one embodiment, Disclosure C relates to the use of an anti-IL-8antibody in producing a pharmaceutical composition for inhibitingfacilitation of neutrophil migration in an individual. In oneembodiment, Disclosure C relates to the use of an effective amount of ananti-IL-8 antibody in inhibiting facilitation of neutrophil migration inan individual. In the above-described embodiments, the “individual” ispreferably a human.

In an alternative embodiment, Disclosure C provides a pharmaceuticalcomposition comprising an anti-IL-8 antibody provided herein forexample, for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical composition comprises an anti-IL-8antibodies provided in Disclosure C and a pharmaceutically acceptablecarrier.

An antibody of Disclosure C can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of DisclosureC may be co-administered with at least one additional therapeutic agent.

An antibody of Disclosure C (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, forexample, by injections such as intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.Various dosing schedules include, without being limited to, single ormultiple administrations over various time-points, bolus administration,and pulse infusion may be contemplated herein.

Preferably an antibody of Disclosure C is formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the pharmaceutical composition, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners. The antibody need not be, but is optionally formulatedwith one or more agents currently used to prevent or treat the disorderin question.

The effective amount of such other agents depends on the amount ofantibody present in the formulation, the type of disorder or treatment,and other factors discussed above. These may be generally used at thesame dosages and via the same administration routes described within thescope of the description of Disclosure C herein, or from 1 to 99% of thedosages described within the scope of the description of Disclosure Cherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

For the prevention or treatment of a disease, the appropriate dose of anantibody of Disclosure C (when used alone or in combination with one ormore other additional therapeutic agents) depends on the type of diseaseto be treated, the type of antibody, the severity and course of thedisease, whether the antibody variant is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and discretion of the attending physician.The antibody is suitably administered to the patient at one time or overa series of treatments. Depending on the type and severity of thedisease, about 1 μg/kg to 15 mg/kg (for example, 0.1 mg/kg to 10 mg/kg)of an antibody can be an initial candidate dose for administration tothe patient, for example, by a single administration or several separateadministrations, or by continuous infusion. One typical daily dose mayrange from about 1 mg/kg to 100 mg/kg or more, depending on the factorsdescribed above. For repeated administrations over several days orlonger, depending on the condition, the treatment may be generallysustained until a desired suppressive effect of disease symptoms isseen. A typical dose of an antibody may fall, for example, in the rangefrom about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of, forexample, about 0.5 mg/kg, for example, 2.0 mg/kg, for example, 4.0mg/kg, or for example, 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, for example, every week or every three weeks (forexample, in such a manner that the patient receives from about two toabout twenty doses, or about six doses of the antibody). It is possibleto administer an initial higher loading dose, followed by one or morelower doses; however, other dosage regimens may be useful. The progressof this therapy can be easily monitored by conventional techniques andassays.

F. Articles of Manufacture

In another aspect of Disclosure C, the disclosure provides articles ofmanufacture comprising materials useful for the treatment, preventionand/or diagnosis of a disorder described above. Such an article ofmanufacture includes a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, and intravenous solution bags. The containers may beformed from various materials such as glass or plastic. Such a containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active ingredient in thecomposition is an antibody of Disclosure C. The label or package insertindicates that the composition is used for treating the condition ofchoice.

Moreover, the article of manufacture may include: (a) a first containerthat comprises a composition comprising an antibody of Disclosure C; and(b) a second container that comprises a composition comprising anadditional cytotoxic agent or a different therapeutic agent. The articleof manufacture in the embodiments of Disclosure C may further include apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively or additionally, the article ofmanufacture may further include, for example, a second (or third)container that comprises a pharmaceutically acceptable buffer such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. The article of manufacture mayfurther include other materials desirable from a commercial perspectiveor a user's standpoint, including other buffers, diluents, filters,needles, and syringes.

Those of ordinary skill in the art can appreciate based on thetechnological common knowledge in the art, that the Disclosure C alsoincludes all combinations of the whole or part of one or more of theentire embodiments described herein, except where there is atechnological inconsistency.

Disclosure A, B, or C

All technical background documents cited herein are incorporated hereinby reference.

As used herein, the phrase “and/or” is understood to include the meaningof combinations of terms before and after the phrase “and/or”, whichinclude all combinations of the terms appropriately linked by thephrase.

While various elements are described herein with terms such as first,second, third, fourth, etc., it is appreciated that the elements are notlimited by such terms. These terms are used only to distinguish anelement from other elements, and it is appreciated that, for example, afirst element could be termed a second element, and similarly, a secondelement could be termed a first element, without departing from thescope of Disclosures A, B, and C.

Unless explicitly stated otherwise or unless there are inconsistenciesin the context, any terms expressed in the singular form herein aremeant to also include the plural form and any terms expressed in theplural form herein are meant to also include the singular form.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the disclosure. Unlessotherwise defined differently, all terms (including technical andscientific terms) used herein are interpreted to have the same meaningas commonly understood by those of ordinary skill in the art to whichDisclosures A, B, and C pertain, and will not be interpreted in anidealized or overly formal sense.

As used herein, the term “comprises” is intended to specify the presenceof described items (members, steps, elements, numbers, etc.), unless thecontext clearly indicates otherwise; and the term does not preclude thepresence of other items (members, steps, elements, numbers, etc.).

Embodiments of the Disclosures A, B, and C are described with referenceto schematic illustrations, which may be exaggerated for clarity.

Unless there are inconsistencies in the context, numerical values usedherein are understood to be values that represent a certain range basedon the common technical knowledge of those of ordinary skill in the art.For example, the expression “1 mg” is understood to be described as“about 1 mg” with certain variations. For example, the expression “1 to5 items” is understood to be described specifically and individually as“1 item, 2 items, 3 items, 4 items, 5 items”, unless there areinconsistencies in the context.

EXAMPLES

Hereinbelow, Disclosures A, B, and C will be specifically described byExamples 1 to 4 and 21 to 23, Examples 5 to 7, 19 and 20, and Examples 8to 19, respectively, but they are not to be construed as being limitedthereto. It is understood that various other embodiments may bepracticed, given the general description provided above.

Example 1 Production of pH-Dependent Human IL-6 Receptor-Binding HumanAntibodies with Increased pI

Fv4-IgG1 disclosed in WO2009/125825 is an antibody that binds to thehuman IL-6 receptor in a pH-dependent manner, and comprises VH3-IgG1(SEQ ID NO:24) as the heavy chain and VL3-CK (SEQ ID NO:32) as the lightchain. To increase the pI of Fv4-IgG1, the variable region of Fv4-IgG1was introduced with amino acid substitutions that decrease the number ofnegatively charged amino acids (such as aspartic acid and glutamicacid), while increasing the positively charged amino acids (such asarginine and lysine). Specifically, VH3(High_pI)-IgG1 (SEQ ID NO:25) wasproduced as a heavy chain with increased pI by substituting glutamicacid at position 16 with glutamine, glutamic acid at position 43 witharginine, glutamine at position 64 with lysine, and glutamic acid atposition 105 with glutamine, according to Kabat numbering, in the heavychain VH3-IgG1. Similarly, VL3(High_pI)-CK (SEQ ID NO:33) was producedas a light chain with increased pI by substituting serine at position 18with arginine, glutamine at position 24 with arginine, glutamic acid atposition 45 with lysine, glutamic acid at position 79 with glutamine,and glutamic acid at position 107 with lysine, according to Kabatnumbering, in the light chain VL3-CK. When introducing the substitutionat position 79 of VL3-CK, modifications that involve substitutingalanine at position 80 with proline and alanine at position 83 withisoleucine were simultaneously introduced, although not with the aim toincrease the pI.

The following antibodies were produced by the method of ReferenceExample 2: (a) Low_pI-IgG1 comprising VH3-IgG1 as the heavy chain andVL3-CK as the light chain; (b) Middle_pI-IgG1 comprising VH3-IgG1 as theheavy chain and VL3(High_pI)-CK as the light chain; and (c) High_pI-IgG1comprising VH3(High_pI)-IgG1 as the heavy chain and VL3(High_pI)-CK asthe light chain.

Next, the theoretical pI for each of the produced antibodies wascalculated using GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION) usingmethods known in the art (see, e.g., Skoog et al., Trends Analyt. Chem.5(4): 82-83 (1986)). The side chains of all cysteines in the antibodymolecule were assumed to form disulfide bonds, and the contribution ofcysteine side chains to pKa was excluded from the calculation.

The calculated theoretical pI values are shown in Table 3. While thetheoretical pI of Low_pI-IgG1 was 6.39, those of Middle_pI-IgG1 andHigh_pI-IgG1 were 8.70 and 9.30, respectively, showing that thetheoretical pI values increased in a stepwise manner.

WO2011/122011 discloses Fv4-IgG1-F11 (hereinafter, referred to asLow_pI-F11) and Fv4-IgG1-F939 (hereinafter, referred to as Low_pI-F939)whose FcRn-mediated uptake into cells has been enhanced by introducingamino acid substitutions into the Fc region of Fv4-IgG1 and conferringFcRn-binding ability under neutral pH conditions. Furthermore,WO2013/125667 discloses Fv4-IgG1-F1180 (hereinafter, referred to asLow_pI-F1180) whose FcγR-mediated uptake into cells has been enhanced byintroducing amino acid substitutions into the Fc region of Fv4-IgG1 toincrease its FcγR-binding ability under neutral pH conditions.Simultaneously, amino acid modification for enhancing the plasmaretention of the antibody by increasing its FcRn binding under theacidic pH condition in the endosomes were introduced intoFv4-IgG1-F1180. The antibodies shown below were produced by increasingthe pI of antibodies containing these novel Fc region variants.

Specifically, VH3-IgG1-F11 (SEQ ID NO:30) and VH3-IgG1-F939 (SEQ IDNO:26) in WO2011/122011, and VH3-IgG1-F1180 (SEQ ID NO:28) inWO2013/125667 were each subjected to substitutions of glutamic acid atposition 16 with glutamine, glutamic acid at position 43 with arginine,glutamine at position 64 with lysine, and glutamic acid at position 105with glutamine, according to Kabat numbering, to produceVH3(High_pI)-F11 (SEQ ID NO:31), VH3(High_pI)-F939 (SEQ ID NO:27), andVH3(High_pI)-F1180 (SEQ ID NO:29), respectively, as heavy chains withincreased pI.

The following antibodies were produced by the method of ReferenceExample 2 using these heavy chains: (1) Low_pI-F939 comprisingVH3-IgG1-F939 as the heavy chain and VL3-CK as the light chain; (2)Middle_pI-F939 comprising VH3(High_pI)-F939 as the heavy chain andVL3-CK as the light chain; (3) High_pI-F939 comprising VH3(High_pI)-F939as the heavy chain and VL3(High_pI)-CK as the light chain; (4)Low_pI-F1180 comprising VH3-IgG1-F1180 as the heavy chain and VL3-CK asthe light chain; (5) Middle_pI-F1180 comprising VH3-IgG1-F1180 as theheavy chain and VL3(High_pI)-CK as the light chain; (6) High_pI-F1180comprising VH3(High_pI)-F1180 as the heavy chain and VL3(High_pI)-CK asthe light chain; (7) Low_pI-F11 comprising VH3-IgG1-F11 as the heavychain and VL3-CK as the light chain; and (8) High_pI-F11 comprisingVH3(High_pI)-F11 as the heavy chain and VL3(High_pI)-CK as the lightchain.

Next, the theoretical pI for each of the produced antibodies wascalculated using GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION) by amethod similar to that described previously. The calculated theoreticalpI values are shown in Table 3. In all novel Fc regionvariant-containing antibodies, the theoretical pI values increased in astepwise manner in the order of Low_pI, Middle_pI, and High_pI.

TABLE 3 Antibody Name Theoretical pI Low_pI-IgG1 6.39 Middle_pI-IgG18.70 High_pI-IgG1 9.30 Low_pI-F939 6.67 Middle_pI-F939 8.70 High_pI-F9399.42 Low_pI-F1180 6.39 Middle_pI-F1180 8.70 High_pI-F1180 9.29Low_pI-F11 6.39 High_pI-F11 9.28

Example 2 Antigen Eliminating Effects of Antibodies with Increased pIthat Show pH-Dependent Binding

(2-1) In Vivo Assay of pI-Adjusted pH-Dependent Human IL-6Receptor-Binding Antibodies

As shown below, in vivo assays were performed using the variouspH-dependent human IL-6 receptor-binding antibodies produced in Example1: Low_pI-IgG1, High_pI-IgG1, Low_pI-F939, Middle_pI-F939, High_pI-F939,Low_pI-F1180, Middle_pI-F1180, and High_pI-F1180.

Soluble human IL-6 receptor (also called “hsIL-6R”) prepared by themethod of Reference Example 3, the anti-human IL-6 receptor antibody,and human immunoglobulin preparation Sanglopor were administeredsimultaneously to human FcRn transgenic mice (B6.mFcRn−/−.hFcRn Tg line32+/+ mouse, Jackson Laboratories; Methods Mol. Biol. 602: 93-104(2010)), and the subsequent in vivo kinetics of the soluble human IL-6receptor were evaluated. A mixed solution containing the soluble humanIL-6 receptor, the anti-human IL-6 receptor antibody, and Sanglopor (atconcentrations of 5 μg/mL, 0.1 mg/mL, and 100 mg/mL, respectively) wasadministered once at 10 mL/kg through the tail vein. Since theanti-human IL-6 receptor antibody was present in sufficient excess ofthe soluble human IL-6 receptor, almost all of the soluble human IL-6receptor was assumed to be bound by the antibody. Blood was collected 15minutes, seven hours, one day, two days, three days, and seven daysafter the administration. The collected blood was immediately subjectedto centrifugation at 4° C. and 15,000 rpm for 15 minutes to obtain theplasma. The separated plasma was stored in a freezer set to −20° C. orbelow until measurements were taken.

(2-2) Measurement of the Soluble Human IL-6 Receptor Concentration inPlasma by the Electrochemiluminescence Method

The soluble human IL-6 receptor concentration in mouse plasma wasmeasured by the electrochemiluminescence method. Samples of solublehuman IL-6 receptor adjusted to concentrations of 250, 125, 62.5, 31.25,15.61, 7.81, or 3.90 pg/mL for the calibration curve, and mouse plasmaassay samples diluted 50-fold or more were prepared, respectively. Thesamples were mixed with a monoclonal anti-human IL-6R antibody (R&D)ruthenium-labeled with SULFO-TAG NHS Ester (Meso Scale Discovery), abiotinylated anti-human IL-6R Antibody (R&D), and Tocilizumab (CASnumber: 375823-41-9) which is a human IL-6 receptor-binding antibody,and then they were allowed to react overnight at 37° C. The finalconcentration of Tocilizumab was adjusted to 333 μg/mL. Then, thereaction solutions were dispensed into a Streptavidin Gold Multi-ARRAYPlate (Meso Scale Discovery). After another hour of reaction at roomtemperature, the reaction solution was washed. Then, immediately afterRead Buffer T(×4) (Meso Scale Discovery) was dispensed into the plate,measurement was carried out using the SECTOR Imager 2400 (Meso ScaleDiscovery). The soluble human IL-6 receptor concentration was calculatedbased on the response in the calibration curve using the analyticalsoftware, SOFTmax PRO (Molecular Devices).

The observed changes in the concentration of the soluble human IL-6receptor in the plasma of human FcRn transgenic mice after theintravenous administration are shown in FIGS. 1, 2, and 3. FIG. 1 showsthe effect of enhancing antigen elimination where the pI of the variableregion was increased in the case of a native IgG1 constant region. FIG.2 shows the effect of enhancing antigen elimination where the pI of thevariable region was increased in an antibody that has been conferredwith the ability to bind to FcRn under a neutral pH condition (F939).FIG. 3 shows the effect of enhancing antigen elimination where the pI ofthe variable region was increased in an antibody whose FcγR-bindingability under a neutral pH condition has been enhanced (F1180).

In all cases, it was shown that by increasing the pI of the antibodies,the rate of antigen elimination by the pH-dependent binding antibodiescan be accelerated. It was also shown that by further conferring anincrease in the binding ability toward FcRn or FcγR under the neutral pHconditions, the rate of antigen elimination can be further acceleratedas compared to when only the pI was increased in the pH-dependentbinding antibodies (comparison of FIG. 1 to FIGS. 2 and 3).

(2-3) In Vivo Infusion Assay of pI-Adjusted pH-Dependent Human IL-6Receptor-Binding Antibodies

An in vivo assay was conducted below using the various pH-dependenthuman IL-6 receptor-binding antibodies produced in Example 1:Low_pI-IgG1, High_pI-IgG1, Low_pI-F11, and High_pI-F11.

An infusion pump (MINI-OSMOTIC PUMP MODEL 2004; alzet) containing asoluble human IL-6 receptor was implanted subcutaneously on the back ofhuman FcRn transgenic mice (B6.mFcRn−/−.hFcRn Tg line 32+/+ mouse,Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)) to producemodel animals whose plasma concentration of the soluble human IL-6receptor was kept constant. Anti-human IL-6 receptor antibodies wereadministered to the model animals, and the in vivo kinetics of theantibodies after the administration were assessed.

Specifically, a monoclonal anti-mouse CD4 antibody obtained by a methodknown in the art was administered once at 20 mg/kg into the tail vein tosuppress the production of neutralizing antibodies potentiallyproducible by the mouse itself against the soluble human IL-6 receptor.Then, an infusion pump containing 92.8 μg/ml of the soluble human IL-6receptor was implanted subcutaneously on the back of the mice. Threedays after implantation of the infusion pump, anti-human IL-6 receptorantibodies were administered once at 1 mg/kg into the tail vein. Bloodwas collected from the mice 15 minutes, seven hours, one day, two days,three or four days, six or seven days, 13 or 14 days, 20 or 21 days, and27 or 28 days after the administration of the anti-human IL-6 receptorantibodies. The collected blood was immediately centrifuged at 15,000rpm and 4° C. for 15 minutes to obtain plasma. The separated plasma wasstored in a freezer at −20° C. or below until measurements were taken.

(2-4) Measurement of the Plasma hsIL-6R Concentration by theElectrochemiluminescence Method

The hsIL-6R concentration in mouse plasma was measured by theelectrochemiluminescence method. Samples of hsIL-6R adjusted to 250,125, 62.5, 31.25, 15.61, 7.81, or 3.90 pg/mL for the calibration curveand mouse plasma assay samples diluted 50-fold or more were mixed with amonoclonal anti-human IL-6R antibody (R&D) ruthenium-labeled withSULFO-TAG NHS Ester (Meso Scale Discovery), a biotinylated anti-humanIL-6R Antibody (R&D), and Tocilizumab, and they were allowed to reactovernight at 37° C. The final concentration of Tocilizumab was adjustedto 333 μg/mL. Then, the reaction solutions were dispensed into aStreptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery). Afteranother hour of reaction at room temperature, the reaction solution waswashed. Then, immediately after Read Buffer T(×4) (Meso Scale Discovery)was dispensed into the plate, measurement was carried out using theSECTOR Imager 2400 (Meso Scale Discovery). The hsIL-6R concentration wascalculated based on the response in the calibration curve using theanalytical software SOFT max PRO (Molecular Devices).

Changes in the measured human IL-6 receptor concentration are shown inFIG. 4. As for both the antibody whose Fc region is that of the nativeIgG1 (High_pI-IgG1) and the antibody which contains the novel Fc regionvariant with enhanced binding toward FcRn under the neutral pHconditions (High_pI-F11), the plasma concentration of the soluble humanIL-6 receptor was decreased in the case a high-pI antibody (also called“High_pI”) is administered as compared to the case a low-pI antibody(also called “Low_pI”) is administered.

Without being bound by a particular theory, results obtained from theseexperiments can also be explained as follows: when the Fc region of theadministered antibody is that of a native IgG antibody, uptake into thecell is thought to take place mainly by non-specific uptake(pinocytosis). Here, since the cell membrane is negatively charged, thehigher the pI of the administered antibody-antigen complex is (i.e., thecharge of the molecule as a whole is inclined toward positive charge),the more readily the complex may approach the cell membrane, and theeasier the nonspecific uptake may take place. When an antibody withincreased pI forms a complex with an antigen, that complex as a wholealso has an increased pI in comparison to a complex formed between theoriginal antibody and the antigen; therefore, uptake into cells may beincreased. Therefore, by increasing the pI of an antibody that showspH-dependent antigen binding, the speed or rate of antigen eliminationfrom the plasma can be further accelerated, and the antigenconcentration in the plasma can be maintained at a lower level.

In these Examples, increase of the pI of the antibody was accomplishedby introducing amino acid substitutions that decrease the number ofnegatively charged amino acids and/or increase the number of positivelycharged amino acids that may be exposed on the surface of the antibodymolecule in the antibody variable region. Those of ordinary skill in theart will understand that effects obtained by such pI increase do notdepend primarily (or substantially) on the type of the target antigen orthe amino acid sequence that constitutes the antibody, but can beexpected to depend on the pI. For example, WO2007/114319 andWO2009/041643 describe the following matters in general terms.

Since the molecular weight of an IgG antibody is sufficiently large, itsmajor metabolic pathway does not involve renal excretion. IgG antibodiesthat have Fc are known to have long half-lives since they are recycledby the salvage pathway of FcRn expressed in cells including theendothelial cells of blood vessels, and IgG is considered to be mainlymetabolized in endothelial cells. More specifically, it is thought thatIgGs that are non-specifically taken up into endothelial cells arerecycled by binding to FcRn, and the molecules that cannot bind FcRn aremetabolized. IgGs whose FcRn-binding ability has been reduced haveshorter blood half-lives, and conversely, the blood half-life can beprolonged by increasing their binding ability toward FcRn. This way,previous methods for controlling the kinetics of IgG in blood involvemodifying Fc to change the binding ability toward FcRn; however, theWorking Examples of WO2007/114319 (mainly, techniques for substitutingamino acids in the FR region) and WO2009/041643 (mainly techniques forsubstituting amino acids in the CDR region) showed that regardless ofthe target antigen type, by modifying the pI of the variable region ofan antibody, its blood half-life can be controlled without modifying theFc. The rate of non-specific uptake of an IgG antibody into endothelialcells is thought to depend on the physicochemical Coulombic interactionbetween the negatively charged cell surface and the IgG antibody.Therefore, it is considered that lowering (increasing) the pI of the IgGantibody and thus reducing (increasing) Coulombic interactions decreases(increases) its non-specific uptake into endothelial cells, andconsequently decreases (increases) its metabolism in endothelial cells,thereby enabling the control of plasma pharmacokinetics. Since theCoulombic interaction between endothelial cells and the cell surface'snegative charge is a physicochemical interaction, this interaction isconsidered not to depend primarily on the antibody-constituting aminoacid sequence per se. Therefore, the methods for controlling plasmapharmacokinetics provided herein are not just applicable to specificantibodies, but they can be widely applied to any polypeptide containingan antibody variable region. Herein, a reduction (an increase) ofCoulombic interactions means a decrease (an increase) of the Coulombicforce represented as an attractive force and/or an increase (a decrease)of the Columbic force represented as a repulsive force.

The amino acid substitutions for accomplishing the above may be a singleamino acid substitution or a combination of multiple amino acidsubstitutions. In some embodiments, a method is provided for introducinga single amino acid substitution or a combination of multiple amino acidsubstitutions into a position(s) exposed on the antibody moleculesurface. Alternatively, the multiple amino acid substitutions introducedmay be positioned conformationally close to each other. The inventorsarrived at the idea that, for example, when substituting amino acidsthat may be exposed on the antibody molecule surface with positivelycharged amino acids (preferably arginine or lysine) or when usingpre-existing positively charged amino acids (preferably arginine orlysine), it may be preferable to further substitute one or more aminoacids that are conformationally proximal to those amino acids (incertain cases, even one or more amino acids buried within the antibodymolecule) with positively charged amino acids to produce, as a result, astate of locally clustered positive charges at conformationally proximalpositions. Here, the definition of “conformationally proximalposition(s)” is not particularly limited, but for example, it may mean astate where a single amino acid substitution or multiple amino acidsubstitutions are introduced within 20A, preferably within 15A, or morepreferably within 10A of one another. Whether the amino acidsubstitution of interest is at a position exposed on the antibodymolecule surface, or whether the amino acid substitution is proximallypositioned can be determined by known methods such as X-raycrystallography.

This way, by noting that the pI is one indicator representing theoverall charge of the molecule, and that charges buried inside theantibody molecule and charges on the antibody molecule surface aretreated without any distinction, the inventors also conceived that byproducing an antibody molecule with broad and comprehensiveconsideration of the effects from charges, which include not only the pIbut also the surface charges and local clustering of charges on antibodymolecules, the speed of antigen elimination from the plasma can befurther accelerated and the antigen concentration in the plasma can bemaintained at even lower levels.

Receptors such as FcRn or FcγR are expressed on the cell membrane, andantibodies that have an enhanced affinity toward FcRn or FcγR underneutral pH conditions are thought to be taken up into cells mainlythrough these Fc receptors. Since the cell membrane is negativelycharged, the administered antibody-antigen complex approaches the cellmembrane more readily when its pI is high (the charge of the molecule asa whole is shifted toward positive charge), and uptake through the Fcreceptor may take place more easily. Therefore, antibodies that have anenhanced affinity towards FcRn or FcγR under neutral pH conditions aswell as an increased pI also show increased uptake into cells through Fcreceptors when they form a complex with antigens. Accordingly, the speedof antigen elimination from the plasma by antibodies that bind toantigens in a pH-dependent manner and have an enhanced affinity towardFcRn or FcγR under neutral pH conditions can be hastened by increasingtheir pIs, and the plasma antigen concentration can be maintained atlower levels.

Example 3 Evaluation of the Extracellular Matrix Binding of pH-DependentBinding Antibodies with Increased pIs

(3-1) Evaluation of the Extracellular Matrix-Binding Ability

The following experiment was carried out to evaluate the effects ofconferring antibodies with the pH-dependent antigen-binding property andfurther modifying the pI on their extracellular matrix-binding ability.

In a manner similar to the method of Example 1, three types ofantibodies with different pI were produced as antibodies that showpH-dependent binding toward the IL-6 receptor: Low_pI-IgG1,Middle_pI-IgG1, and High_pI-IgG1. As ordinary antibodies that do notshow pH-dependent binding to the IL-6 receptor, Low_pI(NPH)-IgG1comprising H54 (SEQ ID NO:34) and L28 (SEQ ID NO:35) andHigh_pI(NPH)-IgG1 comprising H(WT) (SEQ ID NO:36) and L(WT) (SEQ IDNO:37) described in WO2009125825 were produced by the method ofReference Example 2, respectively.

In a manner similar to the method of Example 1, the theoretical pI wascalculated for these antibodies and shown in Table 4. Antibodies that donot show pH-dependent binding to the IL-6 receptor were also shown tohave an increased pI similarly to antibodies that show pH-dependentbinding.

TABLE 4 Antibody Name Theoretical pI Low_pI-IgG1 6.39 Middle_pI-IgG18.70 High_pI-IgG1 9.30 Low_pI(NPH)-IgG1 6.10 High_pI(NPH)-IgG1 9.35(3-2) Evaluation of Antibody Binding to the Extracellular Matrix by theElectrochemiluminescence (ECL) Method

The extracellular matrix (BD Matrigel Basement Membrane Matrix;manufactured by BD) was diluted to 2 mg/mL using TBS (Takara). Thediluted extracellular matrix was dispensed into a MULTI-ARRAY 96 wellPlate, High bind, Bare (manufactured by Meso Scale Discovery:MSD) at 5μL per well, and immobilized overnight at 4° C. Then, 20 mM ACES bufferat pH 7.4 containing 150 mM NaCl, 0.05% Tween 20, 0.5% BSA, and 0.01%NaN₃ was dispensed into the plate for blocking. The antibodies to beevaluated were diluted to 30, 10, and 3 μg/mL using 20 mM ACES buffer atpH 7.4 (ACES-T buffer) containing 150 mM NaCl, 0.05% Tween 20, and 0.01%NaN₃, and then were further diluted using 20 mM ACES buffer at pH 7.4containing 150 mM NaCl, 0.01% Tween 20, 0.1% BSA, and 0.01% NaN₃(Dilution Buffer) to produce a final concentration of 10, 3.3, and 1μg/mL, respectively. The diluted antibody solutions were added to theplate from which the blocking solution was removed, and this was shakenat room temperature for one hour. The antibody solutions were removed,ACES-T buffer containing 0.25% glutaraldehyde was added, and afterletting this stand for 10 minutes, the plate was washed with DPBS(manufactured by Wako Pure Chemical Industries) containing 0.05% Tween20. The antibodies for ECL detection were prepared by sulfo-tagging goatanti-human IgG (gamma) (manufactured by Zymed Laboratories) usingSulfo-Tag NHS Ester (manufactured by MSD). Antibodies for detection werediluted in a dilution buffer to be 1 μg/mL, added to the plate, and thenshaken in the dark at room temperature for one hour. The antibodies fordetection were removed, and a 2-fold diluted solution prepared bydiluting MSD Read Buffer T (4×) (manufactured by MSD) with ultrapurewater was added; and then the amount of luminescence was measured onSECTOR Imager 2400 (manufactured by MSD).

The results are shown in FIG. 5. Antibodies showing pH-dependent bindingas well as antibodies that do not show pH-dependent binding both showedincreased binding toward the extracellular matrix by increasing theirpIs. Furthermore, surprisingly, the effect of improving extracellularmatrix binding by increasing the pI was significant in the antibodieswith pH-dependent antigen binding. In other words, the antibody thatbinds to an antigen in a pH-dependent manner and has high pI(High_pI-IgG1) was found to have the strongest affinity toward theextracellular matrix.

Without being limited to a particular theory, results obtained fromthese experiments can also be explained as follows. The introduction ofhistidine modifications into the antibody variable region is known to beone method for conferring pH-dependent antigen-binding property to anantibody (see e.g., WO2009/125825). Histidine has an imidazoyl group onits side chain, and is uncharged under neutral pH to basic pHconditions, but it is known to be positively charged under acidic pHconditions. Using this property of histidine, by introducing histidineinto the antibody variable region, particularly in the CDR(s) positionedclose to the site of interaction with the antigen, one can change thecharge environment and conformational environment at the site ofinteraction with the antigen between the neutral and acidic pHconditions. Such antibodies can be expected to have an antigen affinitythat changes in a pH-dependent manner. Those of ordinary skill in theart will understand that the effects obtained by such introduction(s) ofhistidine do not primarily (or substantially) depend on the type oftarget antigen or the amino acid sequence that constitutes the antibody,but depend on the site of histidine introduction or the number ofhistidine residues introduced.

WO2009/041643 describes in general terms as follows: protein-proteininteractions consist of hydrophobic interactions, electrostaticinteractions, and hydrogen bonds, and the strength of such binding canusually be represented using a binding constant (affinity) or anapparent binding constant (avidity). pH-dependent binding, where thestrength of binding changes between a neutral pH condition (e.g., pH7.4) and an acidic pH condition (for example, pH 5.5 to pH 6.0), dependson naturally-occurring protein-protein interactions. For example, theaforementioned binding between an IgG molecule and FcRn, which is knownto be a salvage receptor for the IgG molecule, shows strong bindingunder acidic pH conditions and very weak binding under neutral pHconditions. Histidine residues are involved in many of theprotein-protein interactions that change in a pH-dependent manner. Sincethe pKa of a histidine residue is close to 6.0 to 6.5, the state ofproton dissociation in the histidine residue changes between neutral andacidic pH conditions. More specifically, the histidine residue isuncharged and neutral under neutral pH conditions, and functions as ahydrogen atom acceptor; while under acidic pH conditions, it ispositively charged and functions as a hydrogen atom donor. Also in theIgG molecule-FcRn interaction described above, histidine residuespresent on the IgG molecule have been reported to be involved inpH-dependent binding (Martin et al., Mol. Cell. 7(4):867-877 (2001)).

Therefore, substituting a histidine residue for an amino acid residueinvolved in the protein-protein interaction, or introducing a histidineat an interacting site can confer pH dependence to protein-proteininteractions. A similar undertaking has been made for protein-proteininteractions between an antibody and an antigen; and an antibody mutantwith decreased antigen affinity under acidic pH conditions has beensuccessfully obtained by introducing histidine into the CDR sequence ofan anti-egg-white lysozyme antibody (Ito et al., FEBS Lett. 309(1):85-88(1992)). Furthermore, antibodies have been reported which specificallybind to antigens under the low pH of cancer tissues and weakly bind thecorresponding antigen under neutral pH conditions due to introduction ofhistidine in the CDR sequence (WO2003/105757).

Meanwhile, an amino acid residue that is introduced to increase the pIis preferably lysine, arginine, or histidine which have positivelycharged side chains. The standard pKa for these amino acid side chainsis 10.5 for lysine, 12.5 for arginine, and 6.0 for histidine (Skoog etal., Trends Anal. Chem. 5(4):82-83 (1986)). Based on the acid-baseequilibrium theory known in the art, these pKa values mean that in asolution of pH 10.5, 50% of the lysine side chains are positivelycharged and the remaining 50% are uncharged. As the pH of the solutionincreases, the positively charged proportion of lysine side chainsdecreases, and in a solution of pH 11.5 which is 1 pH value higher thanthe pKa for lysine, the positively charged proportion becomesapproximately 9%. On the other hand, as the pH of the solutiondecreases, the positively charged proportion increases, and in asolution of pH 9.5 which is 1 pH value lower than the pKa of lysine, thepositively charged proportion becomes approximately 91%. This theoryworks in a similar manner for arginine and histidine as well. Morespecifically, nearly 100% of lysine or arginine is positively charged ina solution at neutral pH (for example, pH 7.0), whereas approximately 9%of histidine is positively charged. Therefore, while histidine ispositively charged under neutral pH conditions, since that level is lowcompared to lysine or arginine, lysine and arginine are considered asmore favorable amino acids to be introduced for increasing the pI.Furthermore, according to Holash et al. (Proc. Natl. Acad. Sci.99(17):11393-11398 (2002), while the introduction of modifications thatincrease the pI have been considered to be effective as modificationsfor increasing extracellular matrix binding, substitutions introducinglysine or arginine have been considered to be more favorable amino acidmodifications than substitutions introducing histidine for theabove-mentioned reasons.

As first disclosed herein, a surprising synergistic increase in theaffinity of an antibody toward extracellular matrix is possible bycombining the introduction of histidine to confer the pH-dependentantigen-binding property along with a pI-increasing modification of theantibody. In fact, while the pI of High_pI-IgG1 was 9.30, the pI ofHigh_pI(NPH)-IgG1 was 9.35, and therefore the value for High_pI-IgG1 wasslightly lower. Nevertheless, the actual affinity towards extracellularmatrix was clearly stronger for High_pI-IgG1. This shows that theaffinity towards extracellular matrix cannot be necessarily explained byjust the pI level, and can be said to show a synergistic effect fromintroducing a combination of pI-increasing modifications and histidinemodifications. This result is a phenomenon that was both surprising andunexpected.

However, one must note that since the pKa of the amino acid side chainsin a protein are greatly affected by the surrounding environment, theywill not always match the above-mentioned theoretical pKa values. Morespecifically, the above description is presented herein based on generalscientific theory; however, one can easily speculate that there may bemany exceptions in actual proteins. For example, in Hayes et al., J.Biol. Chem. 250(18):7461-7472 (1975), when the pKa of histidinecontained in myoglobin was determined experimentally, while the valuescentered around 6.0, they were reported to vary from 5.37 to 8.05.Naturally, histidine which has a high pKa will be mostly positivelycharged under neutral pH conditions. Therefore, the above-mentionedtheory does not negate the pI-increasing effect of introducing histidineand in the actual three-dimensional structure of the protein. It issufficiently possible that an amino acid modification introducinghistidine, as seen in the case with lysine or arginine, may also toexert an effect of pI increase.

Example 4 pI Increase by One Amino Acid Substitution in the ConstantRegion

Methods for increasing the pI of an antibody that binds to an antigen ina pH-dependent manner by introducing amino acid substitutions into theantibody variable region have been described. In addition, methods forincreasing the pI of an antibody can also be carried out by performingas few as one amino acid substitution in the antibody constant region.

The method of adding one amino acid substitution to the antibodyconstant region to increase pI is not particularly limited, but forexample, it can be performed by the method described in WO2014/145159.As in the case with the variable region, amino acid substitutionsintroduced into the constant region are preferably those that decreasethe number of negatively charged amino acids (such as, aspartic acid orglutamic acid) while increasing the positively charged amino acids(such, as arginine or lysine).

Without limitation, the positions for introducing amino acidsubstitutions in the constant regions are preferably positions whereamino acid side chains may be exposed on the antibody molecule surface.Preferable examples include the method of introducing a combination ofmultiple amino acid substitutions at such positions that may be exposedon the antibody molecule surface. Alternatively, the multiple amino acidsubstitutions introduced are preferably positioned so that they areconformationally close to each other. Furthermore, without limitation,the multiple amino acid substitutions introduced are preferablysubstitutions to positively charged amino acids, so that in certaincases they result in a state where multiple positive charges are presentat conformationally proximal positions. The definition of a“conformationally proximal position” is not particularly limited, butfor example, it may mean a state where a single amino acid substitutionor multiple amino acid substitutions are introduced within 20A,preferably within 15A, or more preferably within 10A of one another.Whether the amino acid substitution of interest is at a position exposedon the antibody molecule surface, or whether the multiple positions ofamino acid substitutions are proximally positioned can be determined byknown methods such as X-ray crystallography.

Furthermore, the method for conferring multiple positive charges atconformationally proximal positions include, in addition to theabove-mentioned methods, a method of using amino acids that areoriginally positively charged in an IgG constant region. Examples ofsuch positively charged amino acid positions include (a) arginine atposition 255, 292, 301, 344, 355, or 416, according to EU numbering; and(b) lysine at position 121, 133, 147, 205, 210, 213, 214, 218, 222, 246,248, 274, 288, 290, 317, 320, 322, 326, 334, 338, 340, 360, 370, 392,409, 414, or 439, according to EU numbering. By performing substitutionwith a positively charged amino acid at a position conformationallyproximal to these positively charged amino acids, it is possible toconfer multiple positive charges at conformationally proximal positions.

(4-1) Production of pH-Dependent Anti-IgE Binding Antibodies

The following three antibodies were produced by the method of ReferenceExample 2 as pH-dependent anti-human IgE antibodies: (1) Ab1, which is aconventional antibody comprising Ab1H (SEQ ID NO:38) as the heavy chainand Ab1L (SEQ ID NO:39) as the light chain; (2) Ab2, which is aconventional antibody comprising Ab2H (SEQ ID NO:40) as the heavy chainand Ab2L (SEQ ID NO:41) as the light chain; and (3) Ab3, which is aconventional antibody comprising Ab3H (SEQ ID NO:42) as the heavy chainand Ab3L (SEQ ID NO:43) as the light chain.

(4-2) Evaluation of pH Dependence in Human IgE Binding

Affinities of Ab1 toward human IgE at pH 7.4 and pH 5.8 were evaluatedas follows. Kinetic analyses of human IgE and Ab1 were performed usingBIACORE T100 (GE Healthcare). Measurements were carried out using thefollowing two buffers as the running buffers: (1) 1.2 mM CaCl₂/0.05%tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 1.2 mM CaCl₂/0.05%tween 20, 20 mM ACES, 150 mM NaCl, pH 5.8.

An appropriate amount of Protein A/G (ACTIGEN) was fixed onto Sensorchip CM4 (GE Healthcare) by the amine coupling method to capture theantibodies of interest. Next, human IgE was made to interact with theantibodies captured onto the sensor chip by injecting a diluted IgEsolution and a running buffer (used as a reference solution). For therunning buffer, either of the buffers (1) and (2) above was used, andhuman IgE was diluted using the respective buffer. To regenerate thesensor chip, 10 mM glycine-HCl at pH 1.5 was used. All measurements werecarried out at 25° C. KD (M) for human IgE was calculated for eachantibody based on the association rate constant ka (1/Ms) anddissociation rate constant kd (1/s), which are kinetic parameterscalculated from the sensorgrams obtained by the measurements. TheBIACORE T100 Evaluation Software (GE Healthcare) was used to calculateeach parameter.

Affinities of Ab2 and Ab3 for human IgE at pH 7.4 and pH 5.8 wereevaluated as follows. The binding activity (dissociation constant KD(M)) of anti-hIgE antibodies toward hIgE were evaluated using BIACORET200 (GE Healthcare). Measurements were carried out using the followingtwo buffers as the running buffers: (1) 1.2 mM CaCl₂/0.05% tween 20, 20mM ACES, 150 mM NaCl, pH 7.4; and (2) 1.2 mM CaCl₂/0.05% tween 20, 20 mMACES, 150 mM NaCl, pH 5.8.

An appropriate amount of a peptide produced by adding biotin to Lyspresent at the C terminus of a chemically synthesized human glypican 3(a.k.a., GPC3) protein-derived peptide (having the amino acid sequenceof (VDDAPGNSQQATPKDNEISTFHNLGNVHSPLK (SEQ ID NO:44))(“biotinylated GPC3peptide”) was added to Sensor chip SA (GE Healthcare) and immobilizedonto the chip by utilizing the affinity between streptavidin and biotin.An appropriate concentration of hIgE was injected and immobilized ontothe chip by capturing of the biotinylated GPC3 peptide. An appropriateconcentration of an anti-hIgE antibody was injected as an analyte, andthis was made to interact with hIgE on the sensor chip. Then, toregenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was injected.All measurements were carried out at 37° C. Association rate constantska (1/Ms) and dissociation rate constants kd (1/s) were calculated byanalyzing the measurement results by curve-fitting using the BIACORET200 Evaluation Software (GE Healthcare), and dissociation constants KD(M) were calculated based on those values.

The results are presented in Table 5. All antibodies, Ab1, Ab2, and Ab3,showed pH-dependent binding toward human IgE, and their affinity at anacidic pH condition (pH 5.8) was shown to be dramatically weakened whencompared with their affinity at a neutral pH condition (pH 7.4).Accordingly, administration of these antibodies to a living animal isexpected to show an effect of accelerating the elimination of human IgEwhich is the antigen.

TABLE 5 Buffer ka Kd KD Antibody Name pH Condition (1/Ms) (1/s) (M) Ab1pH7.4 2.7E+06 6.0E−03 2.3E−09 pH5.8 1.6E+04 3.9E−02 2.4E−06 Ab2 pH7.42.9E+06 4.5E−03 1.5E−09 pH5.8 5.5E+05 5.3E−02 9.7E−08 Ab3 pH7.4 1.6E+067.9E−03 4.9E−09 pH5.8 1.4E+05 3.3E−02 2.3E−07

The theoretical pI values (pIs) for Ab1-Ab3 calculated in a similarmanner to the method of Example 1 are shown in Table 6.

TABLE 6 Antibody Theoretical Name pI Ab1 6.77 Ab2 6.48 Ab3 6.48(4-3) Production of Antibodies with Increased-pI by a Single Amino AcidModification in the Constant Region

Ab1 produced in Example (4-1) is an antibody having native human IgG1 asthe constant region. Ab1H-P600 was produced by modifying the Fc regionof Ab1H, which is the heavy chain of Ab1, through substituting theproline at position 238 according to EU numbering with aspartic acid andsubstituting the serine at position 298 according to EU numbering withalanine. Furthermore, various Fc variants were produced by the method ofReference Example 2 by introducing the various single amino acidsubstitutions indicated in Tables 7-1 and 7-2 into the Fc region ofAb1H-P600, respectively. For all of the Fc variants, Ab1L (SEQ ID NO:39)was used as the light chain. The affinity of these antibodies forhFcγRII2b was comparable to the P600 variant (data not shown).

TABLE 7-1 Variant Amino Acid Mutation Name Added to P600 Biacore ImagingP600 None 1.00 1.00 P828 Q196K 1.27 0.98 P829 S337R 0.17 0.89 P830 L358K1.24 2.35 P831 P387R 3.85 1.30 P836 E345Q 1.85 No Data P837 E345R 1.88No Data P838 D356Q 1.87 No Data P839 D356N 2.17 No Data P840 T359K 2.25No Data P841 N361R 1.86 No Data P842 Q362K 2.37 No Data P843 E380R −0.04No Data P844 E382Q 1.24 No Data P845 E382K 1.38 No Data P846 Q386K 1.71No Data P847 N389K 1.57 No Data P848 S415R 1.38 No Data P849 Q418R 2.21No Data P850 Q419K 2.22 No Data P851 N421R 1.43 1.56 P852 S424K 1.40 NoData P854 L443R 1.93 No Data P905 N384R 1.34 2.36 P906 G385R 1.74 1.12P907 H433R 0.09 3.55 P908 N434R 0.42 1.88 P909 H435R 0.73 0.77 P910L309R 1.73 1.80 P912 T307R 0.24 1.30 P914 D399R 1.72 3.78 P915 S400R0.85 2.01 P917 A327R −0.05 2.49 P918 L328R −0.06 0.00 P919 P329R −0.060.00

TABLE 7-2 P920 A330R 0.01 1.67 P921 P331R −0.07 0.02 P923 Q311R 1.632.88 P924 N315R 2.08 2.66 P925 Y296R −0.05 0.34 P926 Q295R −0.06 0.04P927 E294R 0.25 0.29 P928 E293R 0.20 0.04 P929 P291R 0.47 0.53 P930A287R −0.07 0.00 P931 N286R 0.68 1.27 P932 H285R 1.38 1.96 P934 V282R1.54 1.67 P935 G281R −0.06 2.14 P937 E272R 0.21 0.56 P938 P271R −0.060.04 P939 D270R −0.07 0.01 P940 E269R −0.05 0.01 P941 H268R 0.47 0.44P942 E258R No Data 3.07 P944 T256R No Data 1.49 P945 S254R No Data 5.70P946 I253R No Data 1.05 P947 M252R No Data 0.94 P948 L251R No Data 0.13(4-4) Human FcγRIIb-Binding Assay by BIACORE Using Novel Fc RegionVariant-Containing Antibodies

Fc region variant-containing antibody binding assays between solublehuman FcγRIIb (a.k.a. “hFcγRIIb”) and antigen-antibody complexes wereperformed using BIACORE® T200 (GE Healthcare). Soluble hFcγRIIb wasproduced in the form of a His-tagged molecule using methods known in theart. An appropriate amount of an anti-His antibody was fixed onto Sensorchip CM5 (GE Healthcare) by the amine coupling method using a Hiscapture kit (GE Healthcare) to capture hFcγRIIh. Next, anantibody-antigen complex and a running buffer (as a reference solution)was injected, and interaction was allowed to take place with thehFcγRIIb captured onto the sensor chip. 20 mMN-(2-Acetamido)-2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl₂,and 0.05% (w/v) Tween 20 at pH 7.4 was used as the running buffer, andthe respective buffer was also used to dilute the soluble hFcγRIIb. Toregenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. Allmeasurements were carried out at 25° C. Analyses were performed based onbinding (RU) calculated from sensorgrams obtained by the measurements,and relative values when the binding amount of P600 was defined as 1.00are shown. To calculate the parameters, the BIACORE® T100 EvaluationSoftware (GE Healthcare) was used. The results are shown in Tables 7-1and 7-2 (see the “BIACORE” column in the Tables) and in FIG. 6. SeveralFc variants were shown to have enhanced affinity toward hFcγRIIb fixedon the BIACORE® sensor chip.

While not being restricted to a particular theory, this result can beexplained as follows. The BIACORE® sensor chip is known to be negativelycharged, and this charged state can be considered to resemble the cellmembrane surface. More specifically, the binding of an antigen-antibodycomplex for hFcγRIIb fixed onto the negatively charged BIACORE sensorchip is surmised to resemble the manner in which the antigen-antibodycomplex binds to hFcγRIIb present on a negatively charged cell membranesurface.

The antibodies produced by introducing the pI-increasing modificationinto the Fc region are antibodies in which the charge of the Fc region(constant region) is more positively charged when compared with thosebefore introduction of the modification. Therefore, the Coulombicinteraction between the Fc region (positive charge) and the sensor chipsurface (negative charge) can be considered to have been strengthened bythe pI-increasing amino acid modification. Furthermore, such effects areexpected to take place similarly on the negatively charged cell membranesurface; therefore, they are also expected to show an effect ofaccelerating the speed or rate of uptake into cells in vivo.

From the above results, a ratio of above about 1.2 fold or more for thebinding to hFcγRIIb of a variant when compared to the binding tohFcγRIIb of Ab1H-P600 was considered to have strong charge effect onbinding of an antibody to hFcγRIIb on the sensor chip. Thus, amodification that is expected to yield a charge effect includes, forexample, a modification at position 196, 282, 285, 309, 311, 315, 345,356, 358, 359, 361, 362, 382, 384, 385, 386, 387, 389, 399, 415, 418,419, 421, 424, or 443, according to EU numbering. Preferably themodification is at position 282, 309, 311, 315, 345, 356, 359, 361, 362,385, 386, 387, 389, 399, 418, 419, or 443. The amino acid substitutionintroduced at such position is preferably arginine or lysine. Anotherexample of an amino acid mutation position where such a charge effectcan be expected includes the glutamic acid at position 430 according toEU numbering. The preferred amino acid substitution to be introduced atposition 430 is arginine or lysine which is positively charged, or amonguncharged residues, substitution to glycine or threonine is preferred.

(4-5) Uptake of Fc Region Variant-Containing Antibodies byhFcγRIIb-Expressing Cells

To evaluate the rate of intracellular uptake into an hFcγRIIb-expressingcell line using the produced novel Fc region variant-containingantibodies, the following assay was performed.

An MDCK (Madin-Darby canine kidney) cell line that constitutivelyexpresses hFcγRIIb was produced using known methods. Using these cells,intracellular uptake of antigen-antibody complexes was evaluated.Specifically, pHrodoRed (Life Technologies) was used to label human IgE(antigen) according to an established protocol, and antigen-antibodycomplexes were formed in a culture solution with the antibodyconcentration being 10.8 mg/mL and the antigen concentration being 12.5mg/mL. The culture solution containing the antigen-antibody complexeswas added to culture plates of the above-mentioned MDCK cells whichconstitutively express hFcγRIIb and incubated for one hour, and then thefluorescence intensity of the antigen taken up into the cells wasquantified using InCell Analyzer 6000 (GE healthcare). The amount ofantigen taken up was presented as relative values to the P600 valuewhich is taken as 1.00.

The results are shown in Tables 7-1 and 7-2 (see the “Imaging” column inthe Tables) and in FIG. 7. Strong fluorescence derived from the antigenin the cells was observed in several Fc variants.

While not being restricted to a particular theory, this result can beexplained as follows: the antigen and antibodies added to the cellculture solution form antigen-antibody complexes in the culturesolution. The antigen-antibody complexes bind to hFcγRIIb expressed onthe cell membrane via the antibody Fc region, and are taken up into thecells in a receptor-dependent manner. Ab1 used in this experiment is anantibody that binds to the antigen in a pH-dependent manner; therefore,the antibody can dissociate from the antigen. Since the dissociatedantigen is labeled with pHrodoRed as described earlier, it fluoresces inthe endosomes. Thus, a stronger fluorescence intensity inside the cellcompared to the control is thought to indicate that the uptake of theantigen-antibody complexes into the cells is taking place more quicklyor more frequently.

Here, a ratio of above about 1.05 fold or more of the fluorescenceintensity of the antigen taken up into the cells of the variantscompared to the fluorescence intensity of Ab1H-P600 was considered tohave charge effect on an antigen taken up into the cells. A ratio ofabove about 1.5 fold or more of the fluorescence intensity of theantigen taken up into the cells of the variants compared to thefluorescence intensity of Ab1H-P600 was considered to have a strongcharge effect on an antigen taken up into the cells. Thus, the aboveresults showed that by introducing the pI-increasing modification intothe appropriate position in the Fc region, uptake into cells can beaccelerated as compared to before introduction of the modification. Anamino acid position modification that shows such effect is, for example,position 253, 254, 256, 258, 281, 282, 285, 286, 307, 309, 311, 315,327, 330, 358, 384, 385, 387, 399, 400, 421, 433, or 434, according toEU numbering. Preferably, modification is at position 254, 258, 281,282, 285, 309, 311, 315, 327, 330, 358, 384, 399, 400, 421, 433, or 434,according to EU numbering. An amino acid substitution introduced at sucha position is preferably arginine or lysine. Without limitation, theposition where an amino acid substitution is introduced in the constantregion with the objective of increasing the pI of the antibody may be,for example, the amino acid residue at position 285 according to EUnumbering. Alternatively, other examples may include an amino acidsubstitution of the amino acid residue at position 399 according to EUnumbering.

Example 5 Production of Fc Variants with Enhanced FcRn Binding UnderAcidic pH Conditions for Improving Retention in the Plasma

Under the acidic pH condition in the endosomes, IgG antibodies taken upinto cells are known to be returned to the plasma by binding to FcRn.Therefore, IgG antibodies generally have long plasma half-life comparedto proteins that do not bind to FcRn. Methods that utilize this propertyto enhance plasma retention of antibodies by increasing their FcRnaffinity under acidic pH conditions through the introduction of aminoacid modifications in the antibody Fc region are known. Specifically,methods for improving plasma retention of an antibody by increasing itsaffinity for FcRn under acidic pH conditions through amino acidmodifications, such as the M252Y/S254T/T256E (YTE) modification(Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006)), M428L/N434S(LS) modification (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)),and N434H modification (Zheng et al., Clinical Pharmacology &Therapeutics 89(2):283-290 (2011)) are known.

On the other hand, as described above, Fc variants with increased FcRnaffinity under acidic pH conditions are also known to show undesiredaffinity towards the rheumatoid factor (RF) (WO2013/046704). Therefore,the following examinations were carried out with an objective ofproducing Fc variants that can improve plasma retention with decreasedor substantially no binding to rheumatoid factor.

(5-1) Production of Novel Fc Region Variant-Containing Antibodies

Fc variants with increased FcRn affinity under acidic pH conditions suchas those including the known modifications, YTE, LS, or N434H, andseveral novel Fc variants (F1847m, F1848m, F1886m, F1889m, F1927m, andF1168m) were produced as shown below.

Sequences encoding heavy chains to which amino acid modifications wereintroduced in the Fc region of the heavy chain (VH3-IgG1m) of Fv4-IgG1,which is an anti-human IL-6 receptor antibody, were produced by themethod of Reference Example 1. These heavy chains were used to producethe following antibodies by the method of Reference Example 2: (a)Fv4-IgG1 comprising VH3-IgG1m (SEQ ID NO:46) as the heavy chain andVL3-CK as the light chain; (b) Fv4-YTE comprising VH3-YTE (SEQ ID NO:47)as the heavy chain and VL3-CK as the light chain; (c) Fv4-LS comprisingVH3-LS (SEQ ID NO:48) as the heavy chain and VL3-CK as the light chain;(d) Fv4-N434H comprising VH3-N434H (SEQ ID NO:49) as the heavy chain andVL3-CK as the light chain; (e) Fv4-F1847m comprising VH3-F1847m (SEQ IDNO:50) as the heavy chain and VL3-CK as the light chain; (f) Fv4-F1848mcomprising VH3-F1848m (SEQ ID NO:51) as the heavy chain and VL3-CK asthe light chain; (g) Fv4-F1886m comprising VH3-F1886m (SEQ ID NO:52) asthe heavy chain and VL3-CK as the light chain; (h) Fv4-F1889m comprisingVH3-F1889m (SEQ ID NO:53) as the heavy chain and VL3-CK as the lightchain; (i) Fv4-F1927m comprising VH3-F1927m (SEQ ID NO:54) as the heavychain and VL3-CK as the light chain; and (j) Fv4-F1168m comprisingVH3-F1168m (SEQ ID NO:55) as the heavy chain and VL3-CK as the lightchain.

(5-2) Kinetic Analyses of Binding Toward Human FcRn

Antibodies containing VH3-IgG1m or an above-mentioned variant as theheavy chain and L(WT) (SEQ ID NO:37) as the light chain were produced bythe method of Reference Example 2, and the binding activity toward humanFcRn was evaluated as follows.

Kinetic analyses of human FcRn and each of the antibodies were carriedout using BIACORE T100 (GE Healthcare). An appropriate amount of ProteinL (ACTIGEN) was fixed onto Sensor chip CM4 (GE Healthcare) by the aminecoupling method to capture the antibodies of interest. Next, human FcRnwas made to interact with the antibodies captured on the sensor chip byinjecting a diluted FcRn solution and a running buffer (used as areference solution). For the running buffer, 50 mM sodium phosphate, 150mM NaCl, and 0.05% (w/v) Tween 20 at pH 6.0 was used, and the respectivebuffer was also used to dilute FcRn. To regenerate the sensor chip, 10mM glycine-HCl at pH 1.5 was used. All measurements were carried out at25° C. KD (M) for human FcRn was calculated for each antibody based onthe association rate constant ka (1/Ms) and dissociation rate constantkd (1/s), which are kinetic parameters calculated from sensorgramsobtained by the measurements. The BIACORE T100 Evaluation Software (GEHealthcare) was used to calculate each parameter.

The results are shown in Table 8.

TABLE 8 Variant KD Value (nM) for Name Amino Acid Mutation(s) hFcRn atpH 6.0 IgG1 1382 LS M428L/N434S 116 YTE M252Y/S254T/T256E 148 F1847mN434A/Y436T/Q438R/S440E 367 F1848m N434A/Y436V/Q438R/S440E 295 F1886mM428L/N434A/Y436T/Q438R/S440E 108 F1889m M428L/N434A/Y436V/Q438R/S440E103 F1927m M428L/N434A/Q438R/S440E 125 F1168m N434A/Q438R/S440E 410

Example 6 Evaluation of the Affinity of Fc Region Variant-ContainingAntibodies with Enhanced FcRn Binding Under Acidic pH Conditions Towardthe Rheumatoid Factor

Anti-drug antibodies (ADAs) affect the efficacy and pharmacokinetics oftherapeutic antibodies, and lead to serious side-effects at times;therefore, clinical utility and efficacy of therapeutic antibodies maybe limited by production of ADAs. Many factors influence theimmunogenicity of therapeutic antibodies, and the presence of effector Tcell epitopes is one factor. In addition, the presence of ADA in apatient before administration of the therapeutic antibody (also called“Pre-existing ADA”) may have similar problems. Specifically, in the caseof therapeutic antibodies for patients with autoimmune diseases such asrheumatoid arthritis (RA), rheumatoid factor (RF) which is anautoantibody against human IgG may cause a “pre-existing ADA” problem.Recently, a humanized anti-CD4 IgG1 antibody having an N434H (Asn434His)mutation was reported to induce significant rheumatoid factor binding(Zheng et al., Clinical Pharmacology & Therapeutics 89(2):283-290(2011)). Detailed studies confirmed that the N434H mutation in humanIgG1 increases binding of the rheumatoid factor to the Fc region ofantibodies as compared to that of the parent human IgG1.

The rheumatoid factor is a polyclonal autoantibody against human IgG,and its epitopes in human IgG differ depending on the clone and seem tobe positioned in the CH2/CH3 interface region, and in the CH3 domainthat may overlap with the FcRn-binding epitope. Therefore, mutationsthat increase the binding activity (binding affinity) towards FcRn mayincrease the binding activity (binding affinity) towards specific clonesof the rheumatoid factor.

In fact, regarding Fc with increased affinity for FcRn at acidic pH orneutral pH, not only the N434H modification but many other amino acidmodifications are also known to similarly increase the binding of the Fcto rheumatoid factor (WO2013/046704).

On the other hand, several amino acid modifications that selectivelysuppress the affinity toward the rheumatoid factor while not affectingaffinity toward FcRn have been presented as examples in WO2013/046704,and among them, combinations of two amino acid mutations, namelyQ438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D, have beenindicated. Accordingly, Q438R/S440E was introduced to Fc with novelincreased affinity under acidic pH conditions first disclosed herein toexamine whether binding toward rheumatoid factors can be decreased.

(6-1) Rheumatoid Factor Binding Assay of Fc Region Variant-ContainingAntibodies

A binding assay toward rheumatoid factor was performed by utilizingelectrochemiluminescence (ECL) at pH 7.4 using individual sera(Proteogenex) from 30 RA patients. A 50-fold diluted serum sample, abiotinylated test antibody (1 μg/mL), and a SULFO-TAG NHS Ester (MesoScale Discovery)-labeled test antibody (1 μg/mL) was each mixed andincubated at room temperature for three hours. Thereafter, the mixturewas added to a Streptavidin-coated MULTI-ARRAY 96-well plate (Meso ScaleDiscovery), and the plate was incubated at room temperature for twohours and then washed. After adding Read Buffer T(×4) (Meso ScaleDiscovery) to each well, the plate was immediately set on the SECTORimager 2400 Reader (Meso Scale Discovery), and chemiluminescence wasmeasured.

The results of this assay are shown in FIGS. 8 to 17. Fv4-IgG1 (FIG. 8)which has a native human IgG1 only showed weak binding to the rheumatoidfactor, whereas the existing Fc variants with increased FcRn binding,Fv4-YTE (FIG. 9), Fv4-LS (FIG. 10), and Fv4-N434H (FIG. 11), all showedsignificantly increased rheumatoid factor binding in a number of donors.On the other hand, all novel Fc region variants with increased FcRnbinding, Fv4-F1847m (FIG. 12), Fv4-F1848m (FIG. 13), Fv4-F1886m (FIG.14), Fv4-F1889m (FIG. 15), Fv4-F1927m (FIG. 16), and Fv4-F1168m (FIG.17), showed only weak rheumatoid factor binding, and this showed thatbinding to the rheumatoid factor as a result of modifications toincrease FcRn binding was significantly inhibited.

FIG. 18 shows the average values of rheumatoid factor-binding affinityin the serum of 30 RA patients for each of the variants. All of the sixnew variants showed a lower affinity than the three pre-existingvariants (YTE, LS, and N434H), and they also showed a lower affinitytoward the rheumatoid factor as compared with native human IgG1. Assuch, when considering clinical development of therapeutic antibodieswith improved affinity towards FcRn for autoimmune diseases such asrheumatoid arthritis and the like, the risk associated with therheumatoid factor, which is of concern in existing Fc variants, wassuppressed in the Fc variants first disclosed herein, and accordinglythey may be used more safely than existing known Fc variants.

Example 7 PK Evaluation of the Fc Variants with Increased FcRn BindingUnder Acidic pH Conditions in Cynomolgus Monkeys

In Example 7, the effect of improving plasma retention in cynomolgusmonkeys was evaluated by the following method using novel Fc regionvariant-containing antibodies provided herein whose binding torheumatoid factor was confirmed to be suppressed.

(7-1) Production of Novel Fc Region Variant-Containing Antibodies

The following anti-human IgE antibodies were produced: (a) OHB-IgG1comprising OHBH-IgG1 (SEQ ID NO:56) as the heavy chain and OHBL-CK (SEQID NO:57) as the light chain; (b) OHB-LS comprising OHBH-LS (SEQ IDNO:58) as the heavy chain and OHBL-CK as the light chain; (c) OHB-N434Acomprising OHBH-N434A (SEQ ID NO:59) as the heavy chain and OHBL-CK asthe light chain; (d) OHB-F1847m comprising OHBH-F1847m (SEQ ID NO:60) asthe heavy chain and OHBL-CK as the light chain; (e) OHB-F1848mcomprising OHBH-F1848m (SEQ ID NO:61) as the heavy chain and OHBL-CK asthe light chain; (f) OHB-F1886m comprising OHBH-F1886m (SEQ ID NO:62) asthe heavy chain and OHBL-CK as the light chain; (g) OHB-F1889mcomprising OHBH-F1889m (SEQ ID NO:63) as the heavy chain and OHBL-CK asthe light chain; and (h) OHB-F1927m comprising OHBH-F1927m (SEQ IDNO:64) as the heavy chain and OHBL-CK as the light chain.

(7-2) Monkey PK Assay on Novel Fc Region Variant-Containing Antibodies

The in vivo kinetics of anti-human IgE antibodies in the plasma afteradministration of the anti-human IgE antibodies to cynomolgus monkeyswere evaluated. The anti-human IgE antibody solution was intravenouslyadministered once at 2 mg/kg. Blood collection was performed fiveminutes, (two hours), seven hours, one day, two days, three days, (fourdays), seven days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days,and 56 days after administration. The collected blood was immediatelysubjected to centrifugation at 4° C. and 15,000 rpm for 5 minutes toobtain plasma. The separated plasma was stored in a freezer set to −80°C. or lower until performing the measurements. Eight types of anti-humanIgE antibodies, namely OHB-IgG1, OHB-LS, OHB-N434A, OHB-F1847m,OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927m, were used.

(7-3) Measurement of the Anti-Human IgE Antibody Concentration in thePlasma by ELISA

The concentration of anti-human IgE antibodies in the plasma ofcynomolgus monkeys was measured by ELISA. First, an anti-human IgG kappachain antibody (Antibody Solution) was dispensed into a Nunc-ImmunoPlate, MaxiSorp (Nalge Nunc International) and allowed to standovernight at 4° C. to produce an anti-human IgG kappa chainantibody-immobilized plate. Calibration curve samples having a plasmaconcentration of 640, 320, 160, 80, 40, 20 or 10 ng/mL, and cynomolgusmonkey plasma measurement samples diluted 100-fold or more wereprepared. These calibration curve samples and plasma measurement sampleswere produced such that cynomolgus monkey IgE (product prepared withinthe company) was added at a concentration of 1 ng/mL. Subsequently, thesamples were dispensed into the anti-human IgG kappa chainantibody-immobilized plate, and allowed to stand at room temperature fortwo hours. Then, an HRP-anti human IgG gamma chain antibody (SouthernBiotech) was dispensed, and allowed to stand at room temperature for onehour. Subsequently, a chromogenic reaction was carried out using the TMBChromogen Solution (Life Technologies) as a substrate, and afterstopping the reaction by adding 1N sulfuric acid (Wako), the absorbanceat 450 nm was measured by a microplate reader. The concentration ofanti-human IgE antibody in the monkey plasma was calculated fromabsorbance of the calibration curve using the analytical softwareSOFTmax PRO (Molecular Devices). The measured change in theconcentration of anti-human IgE antibody in the monkey plasma is shownin FIG. 19. From the measured change in the concentration of anti-humanIgE antibody in the monkey plasma, elimination clearance was calculatedby moment analysis using Phoenix WinNonlin Ver. 6.2 (PharsightCorporation). The calculated pharmacokinetic parameters are shown inTable 9. Samples from individuals who were positive for antibodiesagainst the administered sample in plasma were excluded from thecalculation of the change in the anti-human IgE antibody concentrationand clearance in monkey plasma.

TABLE 9 Elimination Clearance of Administered Sample after Anti-HumanIgE Antibody Administration Elimination Clearance Sample Name(mL/day/kg) OHB-IgG1 9.33 OHB-F1847m 2.83 OHB-F1848m 4.02 OHB-F1886m1.92 OHB-F1889m 2.39 OHB-F1927m 1.51 OHB-LS 1.80 OHB-N434A 4.36(7-4) Measurement of Antibodies Against the Administered Samples inPlasma by the Electrochemiluminescence Method

Antibodies in monkey plasma against the administered samples weremeasured by an electrochemiluminescence method. An administered samplethat was ruthenium-labeled using SULFO-TAG NHS Ester (Meso ScaleDiscovery), an administered sample that was biotinylated using EZ-LinkMicro Sulfo-NHS-Biotinylation Kit (Pierce), and a cynomolgus monkeyplasma measurement sample were mixed in equal amounts, and were left tostand overnight at 4° C. The samples were added to a MULTI-ARRAY 96-wellStreptavidin Gold Plate (Meso Scale Discovery), then allowed to react atroom temperature for two hours, and washed. Then, immediately after ReadBuffer T(×4) (Meso Scale Discovery) was dispensed into the plate,measurements were carried out using SECTOR Imager 2400 (Meso ScaleDiscovery).

As a result, all of the novel Fc variants were confirmed to show greatlyimproved plasma retention in comparison to the Fc region of native IgG1.

(7-5) Mouse PK Assay on Fc Variants

The following experiment was carried out to compare F1718, which is anFc variant described in WO2013/046704, and F1848m, which is an Fcvariant newly discovered this time, as Fc variants for increasing FcRnbinding at acidic pH.

Sequences encoding heavy chains into which amino acid modifications wereintroduced into the Fc region of the heavy chain (VH3-IgG1) ofFv4-IgG1(an anti-human IL-6 receptor antibody), were produced by themethod of Reference Example 1. Using these heavy chains, the followingantibodies were produced by the method of Reference Example 2: (a)Fv4-IgG1 comprising VH3-IgG1 as the heavy chain and VL3-CK as the lightchain; and (b) Fv4-F1718 comprising VH3-F1718 (SEQ ID NO:65) as theheavy chain and VL3-CK as the light chain.

The above-mentioned anti-human IL-6 receptor antibodies wereadministered once at 1 mg/kg into the tail vein of human FcRn transgenicmice (B6.mFcRn−/−.hFcRn Tg line 32+/+ mouse; Jackson Laboratories,Methods Mol. Biol. 602:93-104 (2010). Blood was collected 15 minutes,seven hours, one day, two days, three days, seven days, 14 days, 21days, and 28 days after administration of the anti-human IL-6 receptorantibodies. The collected blood was immediately centrifuged at 15,000rpm and 4° C. for 15 minutes to obtain plasma. The separated plasma wasstored in a freezer at −20° C. or below until measurements were taken.

(7-6) Measurement of the Anti-Human IL-6 Receptor Antibody Concentrationin Plasma by ELISA

The concentration of anti-human IL-6 receptor antibodies in the mouseplasma was measured by ELISA. First, an Anti-Human IgG (gamma-chainspecific) F(ab′)₂ Fragment of Antibody (SIGMA) was dispensed into aNunc-Immuno Plate, MaxiSorp (Nalge nunc International) and allowed tostand overnight at 4° C. to produce an anti-human IgG immobilized plate.Calibration curve samples containing an anti-human IL-6 receptorantibody at a plasma concentration of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025,or 0.0125 μg/mL and mouse plasma measurement samples diluted 100-fold ormore were each prepared. 200 μL of 20 ng/mL soluble human IL-6 receptorwas added to 100 μL of the calibration curve samples or the plasmameasurement samples, and then the mixed solutions were allowed to standfor one hour at room temperature. Subsequently, the mixed solutions weredispensed into each well of the anti-human IgG-immobilized plate, andthe plate was allowed to stand for one hour at room temperature. Then, aBiotinylated Anti-Human IL-6R Antibody (R&D) was added to react for onehour at room temperature. Subsequently, Streptavidin-PolyHRP80(Stereospecific Detection Technologies) was added to react for one hourat room temperature, and chromogenic reaction of this reaction solutionwas carried out using TMB One Component HRP Microwell Substrate (BioFXLaboratories) as a substrate. After stopping the reaction by adding 1 Nsulfuric acid (Showa Chemical), the absorbance at 450 nm of the reactionsolution in each well was measured on a microplate reader. The antibodyconcentration in mouse plasma was calculated from the absorbance of thecalibration curve using the analytical software SOFTmax PRO (MolecularDevices).

The results are shown in FIG. 20. F1718, which is an Fc variant forincreasing FcRn binding at acidic pH described in WO2013/046704, did notshow any effect of prolonging antibody PK, but showed plasma retentionequivalent to that of native IgG1.

F1718 has four mutations, namely N434Y/Y436V/Q438R/S440E, introduced inthe Fc region. By contrast, F1848m, first disclosed herein, has beenintroduced with four mutations, namely N434A/Y436V/Q438R/S440E. The onlydifference between the amino acid mutations introduced in these twotypes of Fc's is that the amino acid mutation introduced at position 434according to EU numbering is Y (tyrosine) in F1718 and A (alanine) inF1848m. In Example (7-2), F1848m showed improved plasma retentioncompared to that of the native IgG1, whereas F1718 did not show anyimprovement in plasma retention. Therefore, without limitation, thissuggests that A (alanine) is preferred as the amino acid mutation to beintroduced at position 434 for improving plasma retention.

(7-7) Predicted Immunogenicity Score of Fc Variants

Generation of anti-drug antibodies (ADA) influences the efficacy andpharmacokinetics of therapeutic antibodies, and brings about seriousside effects in some cases; and therefore, clinical utility and drugefficacy of therapeutic antibodies may be limited by the generation ofADA. The immunogenicity of therapeutic antibodies is known to beaffected by many factors, and in particular, the importance of effectorT cell epitopes carried by the therapeutic antibodies in particular hasbeen reported many times.

In silico tools for predicting T cell epitopes such as Epibase (Lonza),iTope/TCED (Antitope), and EpiMatrix (EpiVax) have been developed. Usingthese in silico tools, T cell epitopes in each of the amino acidsequences can be predicted (Walle et al., Expert Opin. Biol. Ther.7(3):405-418 (2007)), and the potential immunogenicity of therapeuticantibodies can be evaluated.

EpiMatrix was used to calculate the immunogenicity scores of evaluatedFc variants. EpiMatrix is a system for predicting the immunogenicity ofa protein of interest by automatically designing sequences of peptidefragments by sectioning the amino acid sequence of the protein to bepredicted for its immunogenicity by nine amino acids, and thencalculating their ability to bind eight major MHC Class II alleles(DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101,DRB1*1301, and DRB1*1501) (Clin. Immunol. 131(2): 189-201 (2009)).

F1718 and F1756 (N434Y/Y436T/Q438R/S440E) described in WO2013/046704contain a N434Y mutation. In contrast, newly disclosed F1848m and F1847mcontain a N434A mutation.

The immunogenicity scores of these four variants, namely F1718, F1848m,F1756 and F1847m, which were calculated as described above, are shown inthe “EpiMatrix Score” column of Table 31. Furthermore, regarding theEpiMatrix Scores, immunogenicity scores corrected for the Tregitopecontent are shown in the “tReg Adjusted Epx Score” column. Tregitope isa peptide fragment sequence present in large amounts mainly innaturally-occurring antibody sequences, and is a sequence considered toinhibit immunogenicity by activating suppressive T cells (Treg).

TABLE 31 Protein EpiMatrix tReg Adjusted Sequence Mutations Score EpxScore F1718 N434Y/Y436V/Q438R/S440E −10.97 −32.64 F1848mN434A/Y436V/Q438R/S440E −15.38 −37.06 F1756 N434Y/Y436T/Q438R/S440E−14.05 −35.73 F1847m N434A/Y436T/Q438R/S440E −18.4 −40.08

According to these results, both the “EpiMatrix Score” and the “tRegAdjusted Epx Score” showed that the immunogenicity scores of N434Avariants F1848m and F1847m were decreased as compared to that of N434Yvariants. This suggests that A (alanine) is preferred as the amino acidmutation to be introduced at position 434 for the lower immunogenicityscores.

Example 8 Production of Humanized Anti-Human IL-8 Antibodies

(8-1) Production of the Humanized Anti-Human IL-8 Antibody hWS-4

Humanized anti IL-8 antibodies disclosed in U.S. Pat. No. 6,245,894 bindto human IL-8 (hIL-8) and block its physiological function. Modifiedhumanized anti-IL-8 antibodies can be produced by combining the variableregion sequences of the heavy and light chains disclosed in U.S. Pat.No. 6,245,894 with virtually any of the various known human antibodyconstant region sequences. Thus, the human antibody constant regionsequences of these modified antibodies are not particularly limited, butnative human IgG1 sequences or native human IgG4 sequences may be usedas the heavy chain constant regions, and native human Kappa sequencescan be used as the light chain constant region sequence.

From among the humanized IL-8 antibodies disclosed in U.S. Pat. No.6,245,894, the coding sequence of hWS4H-IgG1 (SEQ ID NO:83) was combinedthe heavy chain variable region RVHg and the native human anti-IgG1sequence for the heavy chain constant region was produced by the methodof Reference Example 1. Furthermore, the coding sequence of hWS4L-k0MT(SEQ ID NO:84) which was combined the light chain variable region RVLaand the native human Kappa sequence for the light chain constant regionwas produced by the method of Reference Example 1. An antibody which wascombined the above heavy chain and light chain was produced, and wasnamed the humanized WS-4 antibody (hereinafter, hWS-4).

(8-2) Production of Humanized Anti-Human IL-8 Antibody Hr9

A new humanized antibody was produced using human consensus frameworksequences that are different from the FRs used in hWS-4.

Specifically, a hybrid sequence of VH3-23 and VH3-64 was used as theheavy chain FR1, a sequence seen in VH3-15 and VH3-49 was used as FR2, asequence seen in VH3-72 was used as FR3 (provided that 82a according toKabat numbering is excluded), and a sequence seen in JH1 was used asFR4. These sequences were linked to the CDR sequences of the hWS-4 heavychain to produce Hr9-IgG1 (SEQ ID NO:85), a novel humanized antibodyheavy chain.

Next, two types of antibodies were produced, namely, hWS-4 havinghWS4H-IgG1 as the heavy chain and hWS4L-k0MT as the light chain, and Hr9having Hr9-IgG1 as the heavy chain and hWS4L-k0MT as the light chain.Within the scope of Disclosure C described herein, when referring to thelight chain in particular, Hr9 is written as Hr9/hWS4L. The antibodieswere expressed using FreeStyle 293F cells (Invitrogen) according to theprotocol attached to the product. Antibodies were purified from theculture supernatant by the method of Reference Example 2. As a result,antibodies were obtained in the amounts shown in Table 11. Surprisingly,the expression level of Hr9 was approximately 8 times the expressionlevel of hWS-4.

TABLE 11 Antibody Yield per 1 mL Medium (μg) hWS-4 6.4 Hr9 50(8-3) Human IL-8-Binding Activities of hWS-4 and Hr9

Binding affinities of hWS-4 and Hr9 towards human IL-8 were determinedas follows using BIACORE T200 (GE Healthcare).

A running buffer having the composition of 0.05% tween 20, 20 mM ACES,and 150 mM NaCl (pH 7.4) was used. An appropriate amount of Protein A/G(PIERCE) was immobilized onto Sensor chip CM4 (GE Healthcare) by theamine coupling method and the antibody of interest was captured. Next,human IL-8 was made to interact with the antibody captured on the sensorchip by injecting a diluted human IL-8 solution and a running buffer(used as a reference solution). For the running buffer, the solutionhaving the above-described composition was used, and this buffer wasalso used to dilute human IL-8. To regenerate the sensor chip, 10 mMglycine-HCl at pH 1.5 was used. All measurements were carried out at 37°C. KD (M) of each antibody for human IL-8 was calculated based on theassociation rate constant kon (1/Ms) and dissociation rate constant koff(1/s), which are kinetic parameters calculated from sensorgrams obtainedby the measurements. The BIACORE T200 Evaluation Software (GEHealthcare) was used to calculate each parameter.

The results are shown in Table 12. hWS-4 and Hr9 were confirmed to haveequivalent binding affinities toward human IL-8.

TABLE 12 Antibody kon koff KD Name (1/Ms) (1/s) (M) hWS-4 9.74E+052.03E−04 2.09E−10 Hr9 1.11E+06 2.17E−04 1.95E−10

For development of antibody pharmaceuticals, the production level ofantibody molecules is an important factor, and generally, a highproduction level is desirable. It is particularly notable that from theabove-mentioned examination, a more appropriate human consensusframework-derived sequence was selected for combination with the HVRsequence of hWS-4, and yielded Hr9 which had an improved productionlevel while maintaining the binding affinity toward human IL-8.

Example 9 Generation of Antibodies with pH-Dependent IL-8 Affinity

(9-1) Production of Hr9-Modified Antibodies for Conferring pH Dependency

Studies were carried out with the objective of conferring pH-dependentIL-8 affinity to the Hr9 antibody obtained in Example 8.

While not being bound by particular theory, antibodies havingpH-dependent affinity towards IL-8 may show the following behavior invivo. The antibodies administered to a living organism can bind stronglyto IL-8 in an environment where neutral pH is maintained (for example,in plasma), and block its function. A portion of such IL-8/antibodycomplexes are taken up into cells by nonspecific interaction with thecell membrane (pinocytosis) (hereinafter, referred to as non-specificuptake). Under the acidic pH conditions in the endosomes, the bindingaffinities of the: aforementioned antibodies toward IL-8 become weak,and therefore the antibodies dissociate from IL-8. Then, the antibodiesthat dissociated from IL-8 can return to the outside of the cell viaFcRn. The aforementioned antibodies that returned to the outside of thecell (into the plasma) in this manner can bind again to another IL-8 andblock its function. Antibodies having pH-dependent affinity towards IL-8are thought to be capable of binding to IL-8 multiple times by theabove-mentioned mechanism.

In contrast, an antibody that does not have the property possessed bythe aforementioned antibody, an antibody molecule is capable ofneutralizing an antigen only once, but cannot neutralize the antigenmultiple times. Generally, since an IgG antibody has two Fabs, a singleantibody molecule can neutralize two molecules of IL-8. On the otherhand, antibodies which can bind to IL-8 multiple times could bind toIL-8 any number of times as long as they stay in the living body. Forexample, a single molecule of a pH-dependent IL-8-binding antibody thatis taken up into cells ten times since being administered until beingeliminated can neutralize a maximum of 20 molecules of IL-8. Therefore,an antibody that can bind multiple times to IL-8 has the advantage ofbeing able to neutralize several IL-8 molecules even with a small amountof the antibody. From another viewpoint, an antibody that can bindmultiple times to IL-8 has the advantage of being able to maintain astate of being able to neutralize IL-8 for a longer period of time thanwhen the same amount of antibody which does not have the propertypossessed is administered. From yet another viewpoint, an antibody thatcan bind multiple times to IL-8 has the advantage of being able to blockthe biological activity of IL-8 more strongly than when the same amountof an antibody which does not have the property possessed isadministered.

To achieve these advantages, amino acid modifications, mainly histidine,were introduced into the variable regions of Hr9-IgG1 and WS4L-k0MT withthe objective of producing antibodies that can bind to IL-8 multipletimes. Specifically, the variants shown in Table 13 were produced by themethods of Reference Examples 1 and 2.

Notations such as “Y97H” indicated in Table 13 show the position wherethe mutation is introduced as defined by Kabat numbering, the amino acidbefore introduction of the mutation, and the amino acid afterintroduction of the mutation. Specifically, when denoted as “Y97H”, itshows that the amino acid residue at position 97 according to Kabatnumbering has been substituted from Y (tyrosine) to H (histidine).Furthermore, when a combination of multiple amino acid substitutions isintroduced, it is written in a manner such as “N50H/L54H”.

TABLE 13 Antibody Mutation Introduced Mutation Introduced Name intoHeavy Chain into Light Chain Hr9/WS4L None None Hr9/L16 None L54HH89/WS4L Y97H None H89/L12 Y97H N50H H89/L16 Y97H L54H(9-2) pH-Dependent IL-8 Affinity

The human IL-8-binding affinity of the antibodies produced in Example9-1 was determined as described below using BIACORE T200 (GEHealthcare). The following two running buffers were used: (1) 0.05%tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 0.05% tween 20, 20 mMACES, 150 mM NaCl, pH 5.8.

An appropriate amount of Protein A/G (PIERCE) was immobilized ontoSensor chip CM4 (GE Healthcare) by the amine coupling method and theantibodies of interest was captured. Next, human IL-8 was made tointeract with the antibodies captured on the sensor chip by injecting adiluted human IL-8 solution and a running buffer (used as a referencesolution). For the running buffer, any of the above-mentioned solutionswas used, and the respective buffers were also used to dilute humanIL-8. To regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 wasused. All measurements were carried out at 37° C. KD (M) of eachantibody for human IL-8 was calculated based on the association rateconstant kon (1/Ms) and dissociation rate constant koff (1/s), which arekinetic parameters calculated from sensorgrams obtained by themeasurements. The BIACORE T200 Evaluation Software (GE Healthcare) wasused to calculate each parameter.

The results are shown in Table 14-1. First, compared to Hr9, Hr9/L16which contains a L54H modification in the light chain had a slightlyenhanced human IL-8-binding affinity at neutral pH (pH 7.4) but alowered human IL-8-binding affinity at acidic pH (pH 5.8). On the otherhand, anti-IL-8 antibodies (H89/WS4L, H89/L12, and H89/L16) produced bycombining various light chains with H89 containing the Y97H modificationin the heavy chain all showed a decreased human IL-8-binding affinity atacidic pH as well as a decreased human IL-8-binding affinity at neutralpH.

TABLE 14-1 Antibody kon Ratio koff Ratio KD Ratio Name pH kon (1/Ms)koff (1/s) KD (M) (pH7.4/pH5.8) (pH5.8/pH7.4) (pH5.8/pH7 4) Hr9 pH 7.48.59E+05 2.11E−04 2.46E−10 2.7 2.2 5.9 (Hr9/WS4L) pH 5.8 3.23E+054.69E−04 1.45E−09 Hr9/L16 pH 7.4 8.90E+05 9.57E−05 1.08E−10 22.8 2.146.8 pH 5.8 3.91E+04 1.97E−04 5.04E−09 H89/WS4L pH 7.4 8.51E+05 7.65E−048.99E−10 5.2 9.5 49.8 pH 5.8 1.62E+05 7.27E−03 4.48E−08 H89/L12 pH 7.45.95E+05 2.48E−04 4.17E−10 5.0 14.2 71.0 pH 5.8 1.19E+05 3.52E−032.96E−08 H89/L16 pH 7.4 6.02E+05 4.21E−04 6.99E−10 5.0 10.0 50.3 pH 5.81.20E+05 4.22E−03 3.51E−08 H89/L63 pH 7.4 5.37E+05 1.13E−04 2.10E−10 2.118.7 38.3 pH 5.8 2.62E+05 2.10E−03 8.04E−09 H89/L118 pH 7.4 5.80E+052.13E−05 3.67E−11 3.2 180.3 585.0 pH 5.8 1.79E+05 3.84E−03 2.15E−08(9-3) Production and Evaluation of Modified Antibodies for Conferring pHDependence

Combinations of promising modifications found in 9-2 and new amino acidmutations were evaluated, and the following combinations were found as aresult.

TABLE 14-2 Antibody Mutation(s) Introduced Mutation(s) Introduced Nameinto Heavy Chain into Light Chain H89/L63 Y97H N50H/L54H H89/L118 Y97HN50H/L54H/Q89K

The variants were produced by the methods of Reference Examples 1 and 2,and the binding affinity towards human IL-8 was evaluated by a methodsimilar to that of Example 9-2.

The results are also shown in Table 14. H89/L63 which has H89-IgG1 (SEQID NO:86) as the heavy chain and L63-k0MT (SEQ ID NO:87) as the lightchain showed a human IL-8-binding affinity at neutral pH (pH 7.4)equivalent to that of Hr9, and a decreased human IL-8-binding affinityat acidic pH (pH 5.8). Specifically, both the koff (dissociation rateconstant) and KD (dissociation constant) of H89/L63 at pH5.8 were higherthan those of Hr9. This means that under the acidic pH condition in theendosomes, H89/L63 has a property of readily releasing human IL-8.

Surprisingly H89/L118, which has H89-IgG1 as the heavy chain andL118-k0MT (SEQ ID NO:88) as the light chain, was found to have anenhanced human IL-8-binding affinity (KD) under neutral pH conditions ascompared to that of Hr9, but a weakened human IL-8-binding affinity (KD)under acidic pH conditions as compared to that of Hr9. Withoutparticular limitation, generally, when antibodies that can bind multipletimes to antigens are used as a pharmaceutical product, the pH-dependentantigen-binding antibodies preferably have a strong binding affinity(small KD) so that they can strongly neutralize the antigens underneutral pH conditions (such as in plasma). On the other hand, theantibodies preferably have a large dissociation rate constant (koff)and/or a weak binding affinity (large KD) so that they can quicklyrelease the antigens under acidic pH conditions (such as in theendosomes). In comparison to Hr9, H89/L118 had acquired favorableproperties in both these neutral pH and acidic pH conditions.

Thus, useful amino acid modifications were identified for Hr9 such asY97H for its heavy chain and N50H/L54H/Q89K for its light chain. Whilenot being limited thereto, it has been shown that pH-dependentIL-8-binding antibodies that are superior as pharmaceuticals could begenerated by introducing a single or a combination of multiple aminoacid modifications selected from these modifications.

While not being bound by a particular theory, it is considered that animportant factor when using a pH-dependent antigen-binding antibody as apharmaceutical is whether or not the antibody administered to the bodycan release the antigen in the endosome. In this regard, a sufficientlyweak binding (large dissociation constant (KD)) under acidic pHconditions or a sufficiently fast dissociation rate (large dissociationrate constant (koff)) is thought to be important. Therefore, it wasexamined in the following experiment whether the KD or koff of H89/L 118obtained by BIACORE is sufficient for dissociating the antigen in theendosome in vivo.

Example 10 Production of High-Affinity Antibodies for Mouse PK Assay

Methods for confirming the effect of an antibody on the rate of humanIL-8 elimination in mice are not particularly limited. In one instance,the method involves administering an antibody in a condition mixed withhuman IL-8 to mice and then comparing the rate of human IL-8 eliminationfrom mouse plasma.

Here, the reference antibody to be used for the mouse PK assay desirablyhas a sufficiently strong binding affinity under both neutral pH andacidic pH conditions. Then, a search for modifications that confer Hr9with high-affinity was conducted, and as a result H998/L63 havingH998-IgG1 (SEQ ID NO:89) as the heavy chain and L63-k0MT as the lightchain was created.

H998/L63 was used to evaluate the human IL-8-binding affinity by amethod similar to that of Example 9-2. The resulting sensorgrams areshown in FIG. 21.

H998/L63 showed a surprisingly slow dissociation rate under both neutralpH and acidic pH conditions, and was shown to have stronger IL-8-bindingaffinity than Hr9. However, it is known that, due to the mechanicallimits of BIACORE, analytical values such as dissociation rate constant(koff) and dissociation constant (KD) cannot be calculated accurately insuch cases where the protein-protein interaction has a slow dissociationrate. As accurate analytical values could not be obtained for H998/L63,its analytical values are not shown here. However, it is confirmed fromthe results of the experiment that H998/L63 has very strong bindingaffinity at both neutral pH and acidic pH, and is suitable as anantibody to be used for comparison in mouse PK assays.

Example 11 Mouse PK Assay Using the pH-Dependent IL-8-Binding AntibodyH89/L118

(11-1) Mouse PK Assay Using H89/L118

The rate of human IL-8 elimination in vivo was evaluated using H89/L118produced in Example 9 and H998/L63 produced in Example 10.

After simultaneous administration of human IL-8 and anti-human IL-8antibodies to mice (C57BL/6J, Charles river), pharmacokinetics of humanIL-8 were evaluated. A mixed solution of human IL-8 and an anti-humanIL-8 antibody (10 μg/mL and 200 μg/mL, respectively) was administered ina single dose at 10 mL/kg to the tail vein. At this time, since asufficiently excessive amount of the anti-human IL-8 antibody is presentwith respect to human IL-8, almost all the human IL-8 is considered tobe bound to the antibody. Blood was collected five minutes, two hours,four hours, seven hours, one day, two days, three days, seven days, 14days, 21 days, and 28 days after the administration. The collected bloodwas immediately centrifuged at 15,000 rpm and 4° C. for 15 minutes toobtain plasma. The separated plasma was stored in a freezer set at −20°C. or below until measurements were taken.

(11-2) Measurement of the Human IL-8 Concentration in Plasma

The human IL-8 concentration in mouse plasma was determined by anelectrochemiluminescence method. First, an anti-human IL-8 antibody(prepared in-house) comprising a mouse IgG constant region was dispensedinto a MULTI-ARRAY 96-well Plate (Meso Scale Discovery), and was allowedto stand at room temperature for one hour. Then, a PBS-Tween solutioncontaining 5% BSA (w/v) was used for blocking at room temperature fortwo hours to prepare an anti-human IL-8 antibody-immobilized plate.Calibration curve samples containing human IL-8 at a plasmaconcentration of 275, 91.7, 30.6, 10.2, 3.40, 1.13, or 0.377 ng/mL andmouse plasma measurement samples diluted 25-fold or more were prepared.The samples were mixed with hWS-4 and allowed to react overnight at 37°C. Subsequently, 50 μL of the mixed solutions were dispensed into eachwell of the anti-human IL-8 antibody-immobilized plate, and the solutionwas stirred at room temperature for one hour. The final concentration ofhWS-4 was adjusted to 25 μg/mL. Then, after one hour of reaction with aBiotin Mouse Anti-Human Igκ Light Chain (BD Pharmingen) at roomtemperature, and then one hour of reaction with SULFO-TAG LabeledStreptavidin (Meso Scale Discovery) at room temperature, Read Buffer T(×1) (Meso Scale Discovery) was dispensed, and measurements wereperformed immediately with SECTOR Imager 2400 (Meso Scale Discovery).The human IL-8 concentration was calculated based on the response in thecalibration curve using the analytical software, SOFT Max PRO (MolecularDevices).

The resulting data on the concentration of human IL-8 in plasma is shownin FIG. 22, and the values of human IL-8 clearance (CL) from mouseplasma are shown in Table 15.

TABLE 15 Human IL-8 CL (mL/d/kg) Antibody Name H998/L63 H89/L118 #1 21.4472.2 #2 27.5 447.2 #3 24.7 476.0 Average (N = 3) 24.5 465.1 StandardDeviation 3.0 15.6

As clear from FIG. 22, in comparison to human IL-8 administeredsimultaneously with H998/L63, human IL-8 administered simultaneouslywith H89/L118 was shown to be eliminated surprisingly quickly from mouseplasma. Furthermore, CL values which quantitatively represent the rateof human IL-8 elimination from mouse plasma indicate that the rate ofhuman IL-8 elimination was increased about 19-fold for H89/L118 ascompared to H998/L63.

Without being bound by a particular theory, the following can bespeculated from the obtained data. Most of the human IL-8 administeredsimultaneously with the antibody binds to the antibody in the plasma andexist in a complexed form. Human IL-8 bound to H998/L63 may exist in anantibody-bound state even under the acidic pH condition in the endosome,due to the antibody's strong affinity. Thereafter, H998/L63 may bereturned to the plasma via FcRn while still in the human IL-8-complexedform; therefore, when this occurs, human IL-8 is also returned to theplasma at the same time. Therefore, most of the human IL-8 taken up intothe cells again may be returned to the plasma. That is, the rate ofelimination of human IL-8 from plasma decreases remarkably when H998/L63is simultaneously administered. On the other hand, as describedpreviously, human IL-8 taken into cells in a form complexed withH89/L118, a pH-dependent IL-8-binding antibody, may dissociate from theantibody under the acidic pH condition in the endosome. Human IL-8dissociated from the antibody would be degraded after being transferredto the lysosome. Therefore, pH-dependent IL-8-binding antibodies cansignificantly accelerate the elimination of human IL-8 as compared to anIL-8-binding antibody such as H998/L63 which has strong binding affinityat both acidic pH and neutral pH.

(11-3) Mouse PK Assay with Increased Dose of H89/L118

Next, an experiment that verifies the effect of varying the dose ofH89/L118 was carried out as follows. After simultaneous administrationof human IL-8 and H89/L118 (2 mg/kg or 8 mg/kg) to mice (C57BL/6J,Charles river), pharmacokinetics of human IL-8 were evaluated. A mixedsolution of human IL-8 (2.5 μg/mL) and an anti-human IL-8 antibody (200μg/mL or 800 μg/mL) was administered to the tail vein in a single doseof 10 mL/kg. At this time, since a sufficiently excessive amount of theanti-human IL-8 antibody is present compared to human IL-8, almost allof the human IL-8 are considered to be bound to the antibody. Blood wascollected five minutes, seven hours, one day, two days, three days,seven days, 14 days, 21 days, and 28 days after the administration. Thecollected blood was immediately centrifuged at 15,000 rpm and 4° C. for15 minutes to obtain plasma. The separated plasma was stored in afreezer set at −20° C. or below until measurements were taken.

Measurement of the human IL-8 concentration in mouse plasma was carriedout by a method similar to that of Example 11-2. The resulting data onthe human IL-8 concentration in plasma is shown in FIG. 23, and thevalues for human IL-8 clearance (CL) from mouse plasma are shown inTable 16.

TABLE 16 Human IL-8 CL (mL/d/kg) Antibody Name H89/L118 H89/L118Antibody Dose 2 mg/kg 8 mg/kg #1 181.2 93.0 #2 237 101.6 #3 247 114.5Average (N = 3) 221.8 103.0 Standard Deviation 35.6 10.8

As a result, it was confirmed that as compared to the group administeredwith 2 mg/kg of H89/L118, the group administered with 8 mg/kg of theantibody had an approximately 2-fold slower rate of human IL-8elimination.

Herein below, the inventors describe contents surmised as one ofpossible factors that bring about the aforementioned results based onthe scientific background, but the contents of the Disclosure C are notlimited to the contents of the following discussion.

Among the antibodies that are returned from inside the endosome into theplasma via FcRn, it is preferable that the proportion of humanIL-8-bound antibodies is low. With the focus on human IL-8 present inthe endosome, it is desirable to have a high proportion of the free formnot bound by an antibody. When human IL-8 is administered together withan antibody that does not have pH-dependent IL-8-affinity, most (nearly100%) of the human IL-8 in the endosome is considered to exist in a formcomplexed with the antibody, and a small amount (close to 0%) isconsidered to be in the free form. On the other hand, when administeredtogether with the pH-dependent IL-8-binding antibody (for exampleH89/L118), a certain proportion of human IL-8 should exist in a freeform in the endosome. Hypothetically, the proportion of free form inthis case can be understood as follows: [proportion of free human IL-8in the endosome (%)]=[free human IL-8 concentration in theendosome]/[total human IL-8 concentration in the endosome]×100.

The proportion of free human IL-8 in the endosome as understood by theabove equation is desirably higher, and for example, 20% is morepreferable than 0%, 40% is more preferable than 20%, 60% is morepreferable than 40%, 80% is more preferable than 60%, and 100% is morepreferable than 80%.

Thus, there is a correlation between the proportion of free human IL-8in the endosome described above and the binding affinity (KD) and/ordissociation rate constant (koff) for human IL-8 at acidic pH. That is,the weaker the binding affinity and/or the greater the dissociation ratefor human IL-8 at acidic pH, the higher the proportion of free humanIL-8 in the endosome. However, in the case of pH-dependent IL-8-bindingantibodies which can make the proportion of free human IL-8 close to100% in the endosome, further weakening the binding affinity and/orincreasing the dissociation rate at acidic pH does not necessarily leadto an effective increase in the proportion of free human IL-8. One caneasily understand that, for example, even if the proportion of freehuman IL-8 is improved from 99.9% to 99.99%, such a degree ofimprovement may not be significant.

Furthermore, according to the general chemical equilibrium theory, whenan anti-IL-8 antibody and human IL-8 coexist and their binding reactionand dissociation reaction have reached an equilibrium, the proportion offree human IL-8 is unambiguously determined by three parameters:antibody concentration, antigen concentration, and dissociation constant(KD). Here, when the antibody concentration is high, when the antigenconcentration is high, or when the dissociation constant (KD) is small,complexes are readily formed and the proportion of free human IL-8decreases. On the other hand, when the antibody concentration is low,when the antigen concentration is low, or when the dissociation constant(KD) is large, complex formation becomes difficult, and the proportionof free human IL-8 increases.

Meanwhile, in this experiment, the rate of elimination of human IL-8when H89/L118 was administered at 8 mg/kg was slower than when theantibody was administered at 2 mg/kg. This therefore suggests that inthe endosome, the proportion of free human IL-8 was decreased whenantibody was administered at 8 mg/kg compared to when the antibody wasadministered at 2 mg/kg. The reason for this decrease may be thatincreasing the antibody dosage by four-fold increased the antibodyconcentration in the endosome, and thereby facilitated formation of theIL-8-antibody complex in the endosome. That is, in the groupadministered with an increased dose of the antibody, the proportion offree human IL-8 in the endosome decreased, and therefore the rate ofelimination of human IL-8 has been decreased. This also suggests thatwhen the antibody is administered at 8 mg/kg, the degree of thedissociation constant (KD) of H89/L118 under acidic pH conditions isinsufficient for bringing free human IL-8 to nearly 100%. Morespecifically, if it is an antibody that has a larger dissociationconstant (KD) (weaker binding) under acidic pH conditions, it mayachieve a state of nearly 100% free IL-8 even when the antibody isadministered at 8 mg/kg, and a rate of human IL-8 elimination equivalentto that when the antibody is administered at 2 mg/kg.

Based on the above, to confirm whether the pH-dependent IL-8-bindingantibody of interest can accomplish a proportion of nearly 100% freehuman IL-8 in the endosome, without being particularly limited, one canverify whether there is room for increasing the degree of theantigen-eliminating effect in vivo or not. For example, one methodcompares the rate of human IL-8 elimination when using a novelpH-dependent IL-8-binding antibody to that when H89/L118 is used, wherethe novel antibody has a weaker binding affinity at acidic pH and/or anincreased dissociation rate at acidic pH compared to that of H89/L118.In case that the aforementioned novel pH-dependent IL-8-binding antibodyshows an equivalent rate of human IL-8 elimination to that for H89/L118,this suggests that the binding affinity and/or dissociation rate ofH89/L118 at acidic pH is already at a level sufficient for achieving aproportion of nearly 100% free human IL-8 in the endosome. On the otherhand, in instances where the aforementioned novel pH-dependentIL-8-binding antibody shows a higher rate of human IL-8 elimination,this suggests that the binding affinity and/or dissociation rate ofH89/L118 at acidic pH has room for improvement.

Example 12 Production and Evaluation of the pH-Dependent IL-8-BindingAntibody H553/L118

(12-1) Production of Antibody H553/L118 Having pH-Dependent IL-8 BindingAbility

Here, the inventors aimed to generate antibodies that have an evenweaker human IL-8-binding affinity under acidic pH conditions and/or agreater dissociation rate than those of H89/L118.

Amino acid modifications, mainly involving histidine, were introducedusing H89/L118 as a base, to produce the modified antibodies shown inTable 17 by a method similar to that of Example 9. Furthermore, thehuman IL-8-binding affinity for these antibodies was determined by amethod similar to that of Example 9-2.

Part of the results is shown in Table 17. The antibody H553/L118comprising H553-IgG1 (SEQ ID NO:90) as the heavy chain and L118-k0MT asthe light chain, and the antibody H496/L118 comprising H496-IgG1 (SEQ IDNO:101) as the heavy chain and L118-k0MT as the light chain were shownto have further increased pH dependency than H89/L118.

TABLE 17 Antibody kon koff KD kon Ratio Koff Ratio KD Ratio Name pH(1/Ms) (1/s) (M) (pH7.4/pH5.8) (pH5.8/pH7.4) (pH5.8/pH7.4) H89/L118 pH7.4 9.45E+05 1.14E−04 1.21E−10 pH 5.8 1.23E+05 3.90E−03 3.18E−08 7.734.2 263.0 H496/L118 pH 7.4 1.29E+06 5.03E−05 3.91E−11 pH 5.8 1.78E+055.47E−03 3.07E−08 7.2 108.6 785.0 H553/L118 pH 7.4 1.15E+06 1.13E−049.76E−11 pH 5.8 6.14E+05 3.05E−02 4.97E−08 1.9 270.7 509.3

In the obtained H553/L118, two amino acid modifications, Y55H and R57P,were introduced into the heavy chain of H89/L118. On the other hand,H496/L118, in which only R57P was introduced into the heavy chain ofH89/L118, has an enhanced binding affinity for human IL-8 at neutral pHbut a hardly changed human IL-8-binding affinity at acidic pH, incomparison to H89/L118. More specifically, the R57P modificationintroduced into H89/L118 is a modification that enhances the humanIL-8-binding affinity only at neutral pH without changing the bindingaffinity at acidic pH. Furthermore, H553/L118 produced by introducingthe Y55H modification into the heavy chain of H496/L118 has a maintainedor slightly enhanced binding affinity at neutral pH, but on the otherhand, a decreased binding affinity at acidic pH in comparison to thoseof H89/L118. That is, introducing a combination of the two amino acidmodifications, Y55H and R57P, into H89/L118 enabled further enhancementof the property of decreasing the binding affinity at acidic pH, whilemaintaining or slightly enhancing the binding affinity at neutral pH.

(12-2) Mouse PK Assay Using H553/L118

Evaluation of the rate of human IL-8 elimination in mice using H553/L118was carried out by a method similar to that of Example 11-2. Theresulting data on the human IL-8 concentration in plasma is shown inFIG. 24, and the values of human IL-8 clearance (CL) from mouse plasmaare shown in Table 18.

TABLE 18 Human IL-8 (mL/d/kg) Antibody Name H89/L118 H89/L118 H553/L118H553/L118 Antibody Dose 2 mg/kg 8 mg/kg 2 mg/kg 8 mg/kg #1 181.2 93.0250 256.6 #2 237 101.6 245 248.4 #3 247 114.5 249 244.1 Average (N = 3)221.8 103.0 248 249.7 Standard Deviation 35.6 10.8 3 6.4

As a result, large differences were not observed between H553/L118 andH89/L118 when the data of mice administered with 2 mg/kg antibody werecompared; however, it was confirmed that H553/L118 accelerates theelimination of human IL-8 by 2.5 fold or so in comparison to H89/L118when the data of mice administered with 8 mg/kg antibody were compared.From another viewpoint, H553/L118 did not show difference in the rate ofhuman IL-8 elimination between 2 mg/kg and 8 mg/kg, and a reduction ofthe antigen elimination rate due to increase of the antibody dose aswith H89/L118 was not observed.

Without particular limitation, one reason why such results were obtainedmay be discussed as follows. H533/L118 showed an equivalent rate ofhuman IL-8 elimination when the antibody was administered at 2 mg/kg andat 8 mg/kg. This can indicate that the proportion of free IL-8 in theendosome can reach a level close to 100%, since the IL-8 binding byH553/L118 at acidic pH is sufficiently weak even under the conditions of8 mg/kg-administration. In other words, this suggests that whileH89/L118 can achieve a maximum human IL-8 elimination effect at a doseof 2 mg/kg, its effects may be weakened at a high dose of around 8mg/kg. On the other hand, H553/L118 can achieve a maximum effect ofeliminating human IL-8 even at a high dose of 8 mg/kg.

(12-3) Stability Evaluation Using H553/L118

H553/L118 was shown to be an antibody that can accelerate theelimination of human IL-8 more remarkably than H89/L118 in mice.However, in order for this antibody to sustain this inhibitory effect onhuman IL-8 for a long period of time in vivo, it is also important thatthe IL-8-neutralizing activity is stably kept (stability inIL-8-neutralizing activity of this antibody) during the period when theadministered antibody is present in vivo (for example, in plasma).Accordingly, the stability of these antibodies in mouse plasma wasevaluated by the following method.

Mouse plasma was collected from the blood of C57BL/6J (Charles River) bya method known in the art. 200 μL it of 200 mM PBS (Sigma, P4417) wasadded to 800 μL of mouse plasma to give 1 mL. Furthermore, sodium azidewas added at a final concentration of 0.1% as an antiseptic. Then, eachantibody (Hr9, H89/L118, and H553/L118) was added to the above-mentionedmouse plasma to a final concentration of 0.2 mg/mL. At this point, aportion of the sample was collected as the initial sample. The remainingsample was stored at 40° C. One week and two weeks after storage, aportion of each sample was collected, and they were used as theone-week-stored sample and the two-week-stored sample. All samples werefrozen at −80° C. and stored until each analysis was performed.

Next, anti-IL-8 antibodies contained in mouse plasma were evaluated fortheir human IL-8-neutralizing activity as follows: CXCR1 and CXCR2 areknown receptors for human IL-8. The PathHunter(r) CHO-K1 CXCR2β-Arrestin cell line (DiscoveRx Co., Cat. #93-0202C2) expresses humanCXCR2, and is a cell line artificially produced so as to emitchemiluminescence when human IL-8-mediated signals are transmitted.While it is not particularly limited, the human IL-8-neutralizingactivity possessed by an anti-human IL-8 antibody can be evaluated usingthis cell. When human IL-8 is added to the culture solution of thecells, a certain amount of chemiluminescence is exhibited in a mannerdependent on the concentration of the added human IL-8. When human IL-8and an anti-human IL-8 antibody are added together to the culturesolution, human IL-8 signal transduction may be blocked upon binding ofthe anti-human IL-8 antibody to human IL-8. As a result,chemiluminescence caused by addition of human IL-8 will be inhibited bythe anti-human IL-8 antibody, and the chemiluminescence will be weakerthan when the antibody is not added, or there will be nochemiluminescence at all. Therefore, as the human IL-8 neutralizingactivity possessed by the antibody becomes stronger, the degree ofchemiluminescence becomes weaker; and as the human IL-8 neutralizingactivity possessed by the antibody becomes weaker, the degree ofchemiluminescence becomes stronger.

This is the same for an antibody that has been added to mouse plasma andstored for a certain period of time. If the neutralizing activity of theantibody does not change due to storage in mouse plasma, the degree ofthe above-mentioned chemiluminescence before and after storage shouldnot change. On the other hand, in the case of an antibody whoseneutralizing activity decreases due to storage in mouse plasma, thedegree of chemiluminescence by use of a stored antibody will increase ascompared to that before storage.

Then, the above-mentioned cell line was used to examine whether theneutralizing activity of an antibody stored in mouse plasma wasmaintained. First, the cell line was suspended in the AssayComplete™Cell Plating 0 Reagent, and then seeded into a 384-well plate at 5000cells/well. One day after starting of the cell culture, an experimentwas performed below for determining the concentration of human IL-8 tobe added. Serially diluted human IL-8 solutions, which contain finalhuman IL-8 concentrations from 45 nM (400 ng/mL) to 0.098 nM (0.1ng/mL), were added to the cell culture solution. Next, a detectionreagent was added according to the protocol of the product, and therelative chemiluminescence level was detected using a chemiluminescencedetector. From this result, reactivity of the cells towards human IL-8was confirmed, and the human IL-8 concentration suitable for confirmingthe neutralizing activity of anti-human IL-8 antibodies was determined.Here, the human IL-8 concentration was set to 2 nM.

Next, the aforementioned anti-human IL-8 antibody-added mouse plasma wasused to evaluate the neutralizing activities of the antibodies containedtherein. Human IL-8 at the concentration determined above and theaforementioned anti-human IL-8 antibody-containing mouse plasma wereadded to the cell culture. The amount of mouse plasma to be added wasdetermined so as to contain stepwise concentrations of the anti-humanIL-8 antibody in the range of 2 μg/mL (13.3 nM) to 0.016 μg/mL (0.1 nM).Next, detection reagents were added according to the product protocol,and the relative chemiluminescence levels were detected using achemiluminescence detector.

Here, relative values for the relative chemiluminescence levels at eachantibody concentration were calculated by defining the average relativechemiluminescence level in wells without addition of human IL-8 andantibody as 0%, and by defining the average relative chemiluminescencelevel in wells that have been added with only human IL-8 but no antibodyas 100%.

The results of human IL-8 inhibition assay using human CXCR2-expressingcells are shown in FIG. 25A, which shows results from the initial sample(without preservative treatment in mouse plasma), FIG. 25B, which showsresults for the samples stored at 40° C. for one week, and FIG. 25C,which shows results for the samples stored at 40° C. for two weeks.

As a result, differences in the human IL-8-neutralizing activity beforeand after storage in mouse plasma were not observed for Hr9 andH89/L118. On the other hand, H553/L118 showed decrease in the humanIL-8-neutralizing activity after two-week storage. Therefore, the humanIL-8-neutralizing activity of H553/L118 readily decreases in mouseplasma as compared to that of Hr9 and H89/L118, and H553/L118 was shownto be an antibody having unstable properties in terms of the IL-8neutralizing activity.

Example 13 Production of Antibodies with Reduced PredictedImmunogenicity Score Using an in Silico System

(13-1) Predicted Immunogenicity Score of Various IL-8-Binding Antibodies

Generation of anti-drug antibodies (ADA) influences the efficacy andpharmacokinetics of therapeutic antibodies, and brings about seriousside effects in some cases; and therefore, clinical utility and drugefficacy of therapeutic antibodies may be limited by the generation ofADA. The immunogenicity of therapeutic antibodies is known to beaffected by many factors, and, there are many reports describing theimportance of effector T cell epitopes in the therapeutic antibodies.

In silico tools for predicting T cell epitopes such as Epibase (Lonza),iTope/TCED (Antitope), and EpiMatrix (EpiVax) have been developed. Usingthese in silico tools, T cell epitopes in each of the amino acidsequences can be predicted (Walle et al., Expert Opin. Biol. Ther.7(3):405-418 (2007)), and the potential immunogenicity of therapeuticantibodies can be evaluated.

Here, EpiMatrix was used to calculate the immunogenicity scores of eachof the anti-IL-8 antibodies. EpiMatrix is a system for predicting theimmunogenicity of a protein of interest by automatically designingsequences of peptide fragments by sectioning the amino acid sequence ofthe protein to be predicted for its immunogenicity by nine amino acids,and then calculating their ability to bind eight major MEW Class IIalleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801,DRB1*1101, DRB1*1301, and DRB1*1501) (De Groot et al., Clin. Immunol.131(2):189-201 (2009)).

The immunogenicity scores of the heavy chains and light chains of eachanti-IL-8 antibody, which were calculated as described above, are shownin the “EpiMatrix Score” column of Table 19. Furthermore, regarding theEpiMatrix Scores, immunogenicity scores corrected for the Tregitopecontent are shown in the “tReg Adjusted Epx Score” column. Tregitope isa peptide fragment sequence present in large amounts mainly in nativeantibody sequences, and is a sequence considered to inhibitimmunogenicity by activating suppressive T cells (Tregs).

Furthermore, regarding these scores, the sum of the scores for the heavyand light chains is shown in the “Total” column.

TABLE 19 Heavy Chain Light Chain Total Antibody EpiMatrix tReg AdjustedEpiMatrix tReg Adjusted EpiMatrix tReg Adjusted Name Score Epx ScoreScore Epx Score Score Epx Score hWS-4 62.44 12.18 22.64 −23.89 85.08−11.71 Hr9 56.52 6.27 22.64 −23.89 79.16 −17.62 H89/L118 57.99 7.74 7.16−39.36 65.15 −31.62 H496/L118 54.13 3.87 7.16 −39.36 61.29 −35.49H553/L118 47.88 −2.37 7.16 −39.36 55.04 −41.73

According to these results, both the “EpiMatrix Score” and the “tRegAdjusted Epx Score” showed that the immunogenicity scores of H89/L118,H496/L118, and H553/L118 were decreased as compared to that of hWS-4,which is a known humanized anti-human IL-8 antibody.

Furthermore, with EpiMatrix, it is feasible to compare the frequency ofADA development predicted for the antibody molecule as a whole byconsidering the heavy-chain and light-chain scores with the actualfrequency of ADA development caused by various commercially availableantibodies. Results of performing such analysis are shown in FIG. 26.Due to system limitations, the notations used in FIG. 26 are “W54” forhWS-4, “HR9” for Hr9, “H89L118” for H89/L118, “H496L118” for H496/L118,and “H553L118” for H553/L118.

As shown in FIG. 26, the frequency of ADA development in humans causedby various commercially available antibodies is known to be 45% forCampath (Alemtuzumab), 27% for Rituxan (Rituximab), and 14% for Zenapax(Daclizumab). On the other hand, while the frequency of ADA developmentpredicted from the amino acid sequence was 10.42% for hWS-4 which is aknown humanized anti-human IL-8 antibody, the frequency of H89/L118(5.52%), H496/L118 (4.67%), or H553/L118 (3.45%) newly identified hereinwere significantly lower in comparison to that of hWS-4.

(13-2) Production of Modified Antibodies with Lowered PredictedImmunogenicity Scores

As described above, the immunogenicity scores of H89/L118, H496/L118,and H553/L118 were lower in comparison to that of hWS-4; however, as isapparent from Table 19, the immunogenicity scores for the heavy chainare higher than those for the light chains, which suggests that there isstill room for improvement in the amino acid sequences of the heavychain in particular from the viewpoint of immunogenicity. Then, a searchwas conducted in the heavy chain variable region of H496 for amino acidmodifications that can decrease the immunogenicity score. As a result ofdiligent search, three variants, H496v1 in which alanine at position 52caccording to Kabat numbering was substituted with aspartic acid, H496v2in which glutamine at position 81 was substituted with threonine, andH496v3 in which serine at position 82b was substituted with asparticacid were found. Furthermore, H1004 that contains all three of thesemodifications was produced.

The results of immunogenicity scores calculated by a method similar tothat of Example 13-1 are shown in Table 20.

TABLE 20 Heavy Chain Light Chain Total Antibody EpiMatrix tReg AdjustedEpiMatrix tReg Adjusted EpiMatrix tReg Adjusted Name Score Epx ScoreScore Epx Score Score Epx Score H496/L118 54.13 3.87 7.16 −39.36 61.29−35.49 H496v1/L118 32.17 −18.08 7.16 −39.38 39.33 −57.44 H496v2/L11845.26 −5.00 7.16 −39.36 52.42 −44.36 H496v3/L118 38.27 −11.98 7.16−39.36 45.43 −51.34 H1004/L118 10.79 −39.47 7.16 −39.36 17.95 −78.83H1004/L395 10.79 −39.47 7.79 −38.74 18.58 −78.21

The three heavy chains, H496v1, H496v2, and H496v3, all of which containa single modification, showed decreased immunogenicity scores incomparison to that of H496. Furthermore, H1004, that contains acombination of three modifications, achieved a remarkable improvement ofthe immunogenicity score.

Here, in addition to L118, L395 was identified as the light chainappropriate for combination with H1004. Therefore, in the calculation ofimmunogenicity scores, both the L118 combination and the L395combination were used. As indicated in Table 20, H1004/L118 andH1004/L395, which are combinations of heavy and light chains, alsoshowed very low immunogenicity scores.

Next, the frequency of ADA development for these combinations waspredicted in a manner similar to Example 13-1. The results are shown inFIG. 27. The notations used in FIG. 27 are “V1” for H496v1/L118, “V2”for H496v2/L118, “V3” for H496v3/L118, “H1004L118” for H1004/L118, and“H1004L395” for H1004/L395.

Surprisingly, H1004/L118 and H1004/L395, which have remarkably loweredimmunogenicity scores, also showed improvement in the predicted valuesfor the frequency of ADA development, and showed a predicted value of0%.

(13-3) Measurement of the IL-8-Binding Affinity of H1004/L395

H1004/L395 which is an antibody comprising H1004-IgG1m (SEQ ID NO:91) asthe heavy chain and L395-k0MT (SEQ ID NO:82) as the light chain wasproduced. The binding affinity of H1004/L395 for human IL-8 was measuredas described below using BIACORE T200 (GE Healthcare).

The following two running buffers were used, and measurements werecarried out at the respective temperatures: (1) 0.05% tween 20, 40 mMACES, 150 mM NaCl, pH 7.4, 40° C.; and (2) 0.05% tween 20, 40 mM ACES,150 mM NaCl, pH 5.8, 37° C.

An appropriate amount of Protein A/G (PIERCE) was immobilized onto theSensor chip CM4 (GE Healthcare) by the amine coupling method and theantibodies of interest were captured. Next, a diluted human IL-8solution or a running buffer (used as a reference solution) was injectedto allow interaction of the antibodies captured onto the sensor chipwith human IL-8. For the running buffer, either one of theabove-mentioned solutions was used, and the respective buffers were alsoused to dilute human IL-8. To regenerate the sensor chip, 25 mM NaOH and10 mM glycine-HCl (pH 1.5) were used. KD (M) of each antibody for humanIL-8 was calculated based on the association rate constant kon (1/Ms)and dissociation rate constant koff (1/s) which are kinetic parameterscalculated from sensorgrams obtained by the measurements. The BIACORET200 Evaluation Software (GE Healthcare) was used to calculate eachparameter.

The measurement results are shown in Table 21. In comparison toH89/L118, H1004/L395, with lowered immunogenicity score, had anequivalent KD for human IL-8 at neutral pH, but increased KD and koff atacidic pH; and it was shown to have the property of dissociating readilyfrom IL-8 in the endosome.

TABLE 21-1 kon Ratio koff Ratio Kd Ratio Antibody kon koff (pH7.4/(pH5.8/ (pH5.6/ Name pH (1/Ms) (1/s) KD (M) pH5.8) pH7.4) pH7.4)H89/L118 pH 7.4 7.51E+05 1.29E−04 1.72E−10 5.8 48.7 283.7 pH 5.81.29E+05 6.28E−03 4.88E−08 H1004/L395 pH 7.4 1.02E+06 1.55E−04 1.51E−103.3 218.1 728.5 pH 5.8 3.06E+05 3.38E−02 1.10E−07

Example 14 Production and Evaluation of the pH-Dependent IL-8-BindingAntibody H1009/L395

(14-1) Production of Various pH-Dependent IL-8-Binding Antibodies

H1004/L395, which has pH-dependent IL-8 binding ability and also alowered immunogenicity score was obtained by the evaluation shown inExample 13. Subsequently, a dedicated investigation was carried out toproduce variants that have these favorable properties as well asstability in mouse plasma.

The following modified antibodies were produced based on H1004/L395 byintroducing various modifications.

TABLE 21-2 Heavy Chain H1004 A52cD/R57P/Q81T/S82bD/Y97H H0932A52cD/G54H/Y55H/R57P/Q81T/S82bD/Y97H H1000D31E/A52cD/G54H/Y55H/R57P/Q81T/S82bD/Y97H H1009A52cD/G54Y/Y55H/R57P/Q81T/S82bD/Y97H H1022A52cD/G54H/Y55H/T56H/R57P/Q81T/S82bD/Y97H H1023A52cD/T56H/R57P/Q81T/S82bD/Y97H H1028A52cD/G54Y/Y55H/T56H/R57P/Q81T/S82bD/Y97H H1029S30D/D31K/A52cD/G54H/Y55H/R57P/Q81T/S82bD/Y97H H1031S30D/D31K/A52cD/G54H/Y55H/T56H/R57P/Q81T/S82bD/ Y97H H1032S30D/D31K/A52cD/T56H/R57P/Q81T/S82bD/Y97H H1037S30D/D31K/A52cD/G54Y/Y55H/T56H/R57P/Q81T/S82bD/ Y97H H1040D31E/A52cD/G54H/Y55H/T56H/R57P/Q81T/S82bD/Y97H H1041D31E/A52cD/T56H/R57P/Q81T/S82bD/Y97H H1046D31E/A52cD/G54Y/Y55H/T56H/R57P/Q81T/S82bD/Y97H H1047S30D/D31K/A52cD/R57P/Q81T/S82bD/Y97H H1048D31E/A52cD/R57P/Q81T/S82bD/Y97H H1049S30D/D31K/A52cD/G54Y/Y55H/R57P/Q81T/S82bD/Y97H H1050D31E/A52cD/G54Y/Y55H/R57P/Q81T/S82bD/Y97H

TABLE 21-3 L395 N50K/L54H/Q89K L442 S31E/N50K/L54H/Q89K

A total of 36 types of antibodies were produced by combining the 18types of heavy chains and two types of light chains described above.Various evaluations were performed on these antibodies as indicatedbelow.

The human IL-8-binding affinities under neutral and acidic pH conditionswere measured in a manner similar to the method of Example 13-3. Amongthe obtained results, KD at pH 7.4, and KD and koff at pH 5.8 are shownin Table 22.

Next, stability in terms of IL-8 binding upon storage of the antibodiesin PBS was evaluated by the method indicated below.

The respective antibodies were dialyzed overnight against DPBS(Sigma-Aldrich), and then the concentration of each of the antibodieswas adjusted to 0.1 mg/mL. At this point, some of the antibody sampleswere collected as initial samples. The remaining samples were stored at50° C. for one week, and then collected as samples for the thermalacceleration test.

Next, BIACORE measurement of the IL-8-binding affinity was carried outas follows using the initial samples and samples for the thermalacceleration test.

The levels of human IL-8 binding to the modified antibodies wereanalyzed using BIACORE T200 (GE Healthcare). Measurements were carriedout at 40° C. by using 0.05% tween 20, 40 mM ACES, and 150 mM NaCl at pH7.4 as the running buffer.

An appropriate amount of Protein A/G (PIERCE) was immobilized onto theSensor chip CM4 (GE Healthcare) by the amine coupling method and theantibodies of interest was captured. Next, a diluted human IL-8 solutionor a running buffer (used as a reference solution) was injected to allowinteraction of the antibodies captured onto the sensor chip with humanIL-8. The running buffer was also used to dilute human IL-8. Toregenerate the sensor chip, 25 mM NaOH and 10 mM glycine-HCl (pH 1.5)were used. The measured binding level of human IL-8 and the amount ofantibodies captured at that binding level were extracted using theBIACORE T200 Evaluation Software (GE Healthcare).

The amount of human IL-8-binding per 1000 RU of the amount of antibodycaptured was calculated for the initial samples and the samples for thethermal acceleration test. Furthermore, the ratio of the humanIL-8-binding level for the initial samples to that for samples of thethermal acceleration test was calculated.

The resulting ratios of IL-8-binding level of the initial samples tothat for samples of the thermal acceleration test are shown in Table 22as well.

TABLE 22 Ratio of IL-8 Binding Amount (Thermal Antibody pH7.4 KD pH5.8KD pH5.8 koff Acceleration/Initial) H0089/L0118 1.7E−10 4.9E−08 6.3E−030.61 H0932/L0395 1.6E−10 1.1E−07 5.7E−02 0.56 H0932/L0442 2.1E−107.9E−08 2.2E−02 0.56 H1000/L0395 1.4E−10 8.9E−08 2.0E−02 0.57H1000/L0442 2.0E−10 7.1E−08 1.7E−02 0.57 H1004/L0395 1.5E−10 1.1E−073.4E−02 0.58 H1004/L0442 2.2E−10 7.7E−08 2.0E−02 0.59 H1009/L03957.1E−11 8.7E−08 1.0E−02 0.64 H1009/L0442 1.1E−10 6.3E−08 6.0E−03 0.64H1022/L0395 2.7E−10 2.9E−07 1.2E+01 0.47 H1022/L0442 3.6E−10 1.8E−072.0E−02 0.46 H1023/L0395 7.6E−11 9.2E−08 1.8E−02 0.54 H1023/L04421.2E−10 7.1E−08 1.7E−02 0.55 H1028/L0395 1.8E−10 2.1E−07 1.0E+01 0.55H1028/L0442 2.4E−10 1.4E−07 1.3E−01 0.56 H1029/L0395 8.6E−11 5.5E−088.0E−03 0.59 H1029/L0442 1.4E−10 4.8E−08 8.5E−03 0.58 H1031/L03951.5E−10 9.9E−08 4.6E−02 0.48 H1031/L0442 2.1E−10 8.9E−08 3.9E−02 0.47H1032/L0395 4.2E−11 5.0E−08 4.1E−03 0.61 H1032/L0442 7.8E−11 4.3E−085.9E−03 0.61 H1037/L0395 9.4E−11 7.0E−08 1.5E−02 0.55 H1037/L04421.3E−10 6.1E−08 1.5E−02 0.57 H1040/L0395 2.6E−10 2.4E−07 4.6E−02 0.44H1040/L0442 3.4E−10 1.4E−07 2.1E+01 0.49 H1041/L0395 8.0E−11 7.1E−081.3E−02 0.55 H1041/L0442 1.2E−10 6.1E−08 1.5E−02 0.56 H1046/L03951.8E−10 1.6E−07 1.2E−02 0.56 H1046/L0442 2.3E−10 1.1E−07 1.2E−02 0.55H1047/L0395 9.5E−11 4.7E−08 6.0E−03 0.65 H1047/L0442 1.5E−10 4.7E−084.6E−03 0.64 H1048/L0395 1.5E−10 9.0E−08 6.4E−03 0.59 H1048/L04422.1E−10 6.7E−08 1.5E−02 0.59 H1049/L0395 2.5E−11 3.8E−08 4.0E−03 0.65H1049/L0442 5.3E−11 3.3E−08 4.5E−03 0.65 H1050/L0395 6.6E−11 7.7E−085.0E−03 0.64 H1050/L0442 9.9E−11 5.4E−08 7.6E−03 0.64

By the above-mentioned examination, H1009/L395 which is an antibodycomprising H1009-IgG1m (SEQ ID NO:92) as the heavy chain and L395-k0MTas the light chain was obtained.

As shown in Table 22, in comparison to H89/L118, H1009/L395 had aslightly enhanced human IL-8-binding affinity at neutral pH, but on theother hand, a decreased binding affinity at acidic pH, that is,pH-dependence had been further strengthened. Furthermore, when exposedto severe conditions such as at 50° C. in PBS, H1009/L395 had a slightlyenhanced stability in IL-8 binding when compared to that of H89/L118.

Accordingly, H1009/L395 was selected as an antibody whose neutralizingactivity in mouse plasma may be stably maintained, while keeping itspH-dependent IL-8 binding ability.

(14-2) Stability Evaluation of H1009/L395

Next, in a manner similar to the method of Example 12-3, it wasevaluated whether the IL-8 neutralizing activity of H1009/L395 is stablymaintained in mouse plasma. Here, H1009/L395-F1886s which will bedescribed in detail later in Example 19 was used. This antibody has thesame variable region as that of H1009/L395, and a constant region havingmodifications that enhance FcRn binding under acidic pH conditions andmodifications for reducing its binding towards FcγR(s) in comparison tothose of the native human IgG1. The variable region of H1009/L395,especially the region around HVR, is responsible for human IL-8-bindingand IL-8-neutralizing activity of this antibody, and modificationsintroduced into the constant region are considered not to affect theseproperties.

Evaluation of the stability in mouse plasma was performed as follows.150 μL of 200 mM phosphate buffer (pH 6.7) was added to 585 μL of mouseplasma. Then, sodium azide was added as an antiseptic at a finalconcentration of 0.1%. Each antibody (Hr9, H89/L118, orH1009/L395-F1886s) was added to the above-mentioned mouse plasma at afinal concentration of 0.4 mg/mL. At this point, a portion of the samplewas collected as the initial sample. The remaining sample was stored at40° C. One week and two weeks after the start of storage, a portion ofeach sample was collected, and they were used as the sample stored forone week and the sample stored for two weeks. All samples were frozen at−80° C. and stored until each analysis was performed.

Measurement of the human IL-8-neutralizing activity was carried outusing human CXCR2-expressing cells by a method similar to that ofExample 12-3. However, the concentration of human IL-8 used to confirmthe neutralizing activity of an anti-human IL-8 antibody this time was1.2 nM.

The results of human IL-8 inhibition assay obtained using theabove-mentioned antibodies with human CXCR2-expressing cells are shownin FIG. 28A, which shows results for the initial sample (without storagetreatment in mouse plasma), FIG. 28B, which shows results for thesamples stored at 40° C. for one week, and FIG. 28C, which shows resultsfor the samples stored at 40° C. for two weeks.

As a result, surprisingly, the human IL-8-neutralizing activity wasmaintained in H1009/L395-F1886s even after it was stored in mouse plasmaat 40° C. for two weeks, and the IL-8-neutralizing activity was morestably maintained than in the case of H553/L118.

(14-3) Mouse PK Assay Using H1009/L395

The rate of human IL-8 elimination by H1009/H395 in mice was evaluatedby the following method. H1009/L395, H553/L118, and H998/L63 were usedas the antibodies. Administration to mice and blood collection, andmeasurement of the human IL-8 concentration in mouse plasma were carriedout by the method shown in Example 11.

The resulting data on the concentration of human IL-8 in plasma areshown in FIG. 29, and the values of human IL-8 clearance (CL) from mouseplasma are shown in Table 23.

TABLE 23 Human IL-8 CL (mL/d/kg) Antibody Name H998/L63 H553/L118H1009/1395 #1 21.4 773.2 705.0 #2 27.5 497.6 777.3 #3 24.7 879.8 737.7Average (N = 3) 24.5 716.9 740.0 Standard Deviation 3.0 197.2 36.2

As a result, the rate of human IL-8 elimination in mice when H1009/L395was administered at 2 mg/kg was equivalent to that of H553/L118, and itwas shown that H1009/L395 achieves nearly 100% free IL-8 in theendosome. The value of clearance (CL) which quantitatively representsthe rate of human IL-8 elimination from mouse plasma was shown to beapproximately 30-fold higher than that of H998/L63.

Without being particularly limited, the effect of increasing the rate ofhuman IL-8 elimination can be understood as follows. Generally, in aliving body where antigens are maintained at nearly constantconcentrations, production rates and elimination rates of antigens willalso be maintained at nearly constant values. When antibodies areadministered under such conditions, even in cases where the antigenproduction rates are not affected, the rates of antigen elimination maychange due to the complex formation of antigen with antibodies.Generally, since the antigen-elimination rate is greater than theantibody-elimination rate, in such cases, the elimination rate ofantigens that have formed complexes with antibodies decreases. When theantigen elimination rate decreases, the antigen concentration in plasmaincreases, but the degree of increase in this case may also be definedby the ratio of the elimination rate when the antigen is present aloneto the elimination rate when the antigen forms a complex. That is, incomparison to the elimination rate when the antigen is present alone, ifthe elimination rate when a complex is formed is decreased to one tenth,the antigen concentration in the plasma of the antibody-administeredorganism may increase up to approximately ten times that before antibodyadministration. Here, clearance (CL) may be used as the eliminationrate. More specifically, increase of the antigen concentration (antigenaccumulation) that takes place after antibody administration to anorganism may be defined by the antigen CL under each of the conditionsbefore antibody administration and after antibody administration.

Here, the presence of an approximately 30-fold difference in CL of humanIL-8 when H998/L63 and H1009/L395 were administered suggests that theremay be an approximately 30-fold difference between the levels ofincrease in the human IL-8 concentration in plasma when these antibodiesare administered to humans. Furthermore, generation of a 30-folddifference in the human IL-8 concentration in plasma indicates thatthere will also be approximately a 30-fold difference in the amount ofantibodies necessary for completely blocking the biological activity ofhuman IL-8 under the respective conditions. That is, in comparison toH998/L63, H1009/L395 can block the biological activity of IL-8 in plasmaat approximately 1/30 of the amount, which is a very small amount ofantibody. Furthermore, when H1009/L395 and H998/L63 are individuallyadministered to humans at the same dose, H1009/L395 will be able toblock the biological activity of IL-8 for a longer period of time withgreater strength. To block the biological activity of IL-8 for a longperiod of time, it is necessary that the IL-8-neutralizing activity isstably maintained. As shown in Example 14, experiments using mouseplasma have elucidated that H1009/L395 can maintain its humanIL-8-neutralizing activity for a long period of time. H1009/L395 whichhas these noteworthy properties was also shown to be an antibody thathas superior effects from the viewpoint of the efficacy in neutralizingIL-8 in vivo.

Example 15 Evaluation of Extracellular Matrix-Binding Using thepH-Dependent IL-8-Binding Antibody H1009/L395

The excellent 30-fold greater effect of H1009/L395 in eliminating humanIL-8 as shown in Example 14 was a surprising effect. It is known thatthe rate of antigen elimination when a pH-dependent antigen-bindingantibody is administered depends on the rate of uptake of theantibody-antigen complex into cells. That is, if the rate of thepH-dependent antigen-binding antibody uptake into cells increases whenan antigen-antibody complex is formed in comparison to when the complexis not formed, the antigen-eliminating effect of the pH-dependentantibody can be increased. Known methods for increasing the rate ofuptake of an antibody into cells include the method of conferring theFcRn-binding ability under neutral pH conditions to an antibody (WO2011/122011), the method for enhancing the binding ability of anantibody towards FcγR(s) (WO 2013/047752), and the method that usespromotion of the formation of complexes containing a polyvalent antibodyand a polyvalent antigen (WO 2013/081143).

However, the above-mentioned technique is not used in the constantregions of H1009/L395. Furthermore, while IL-8 is known to form ahomodimer, human IL-8 bound by H1009/L395 has been found to exist in theform of a monomer because H1009/L395 recognizes the homodimer-formingsurface of human IL-8. Therefore, this antibody will not form polyvalentcomplexes.

More specifically, while the above-mentioned technique is not used forH1009/L395, H1009/L395 showed a 30-fold greater human IL-8-eliminatingeffect.

Then, the inventors carried out the following discussion as a possiblefactor that may bring about the aforementioned properties ofpH-dependent IL-8-binding antibodies represented by H1009/L395. However,the following is only a possibility surmised by the inventors based onthe technical background, and the content of Disclosure C is not limitedto the content of the following discussion.

Human IL-8 is a protein that has a high isoelectric point (pI), and thetheoretical isoelectric point calculated by a known method isapproximately 10. That is, under neutral pH conditions, human IL-8 is aprotein whose charge is shifted towards the positive side. pH-dependentIL-8-binding antibodies represented by H1009/L395 are also proteinswhose charge is shifted towards the positive side, and the theoreticalisoelectric point of H1009/L395 is approximately 9. That is, theisoelectric point of a complex produced by binding of H1009/L395, aprotein that has a high isoelectric point and is originally rich inpositive charges, to human IL-8 which has a high isoelectric point willbe higher than that of H1009/L395 alone.

As shown in Example 3, increasing the isoelectric point of an antibody,which includes increasing the number of positive charges and/ordecreasing the number of negative charges on the antibody, can beconsidered to increase non-specific uptake of the antibody-antigencomplex into cells. The isoelectric point of complex formed between ananti-IL-8 antibody and human IL-8 which has a high isoelectric point ishigher compared to that of the anti-IL-8 antibody alone, and the complexmay be taken up more readily into cells.

As described earlier, affinity for the extracellular matrix is also afactor that may influence uptake into cells. Then, it was examinedwhether there is a difference in extracellular matrix binding between anantibody alone and a complex with a human IL-8-antibody.

Evaluation of the Amount of Antibody Binding to the Extracellular Matrixby the ECL (Electroluminescence) Method

Extracellular Matrix (the BD Matrigel Basement MembraneMatrix/manufactured by BD) was diluted to 2 mg/mL using TBS (Takara,T903). The diluted extracellular matrix was dispensed into theMULTI-ARRAY 96well Plate, High bind, Bare (manufactured by Meso ScaleDiscovery: MSD) at 5 μL per well, and immobilized overnight at 4° C.Then, blocking was performed using 20 mM ACES buffer (pH 7.4) containing150 mM NaCl, 0.05% Tween 20, 0.5% BSA, and 0.01% NaN₃.

The antibodies to be evaluated were prepared as follows. The antibodysamples to be added alone were prepared by diluting each antibody to 9μg/mL using Buffer 1 (20 mM ACES buffer containing 150 mM NaCl, 0.05%Tween 20, and 0.01% NaN₃, at pH 7.4), and then further diluting themusing Buffer2 (20 mM ACES buffer containing 150 mM NaCl, 0.05% Tween 20,0.1% BSA, and 0.01% NaN₃, at pH 7.4) to a final concentration of 3μg/mL.

On the other hand, the antibody samples to be added as a complex withhuman IL-8 were prepared by adding human IL-8 at ten times the molarconcentration of the antibody to an antibody sample, then diluting eachantibody using Buffer-1 so that the antibody concentration became 9μg/mL, respectively, and then further diluting each of them usingBuffer-2 to a final antibody concentration of 3 μg/mL. At this point,the human IL-8 concentration was approximately 0.6 μg/mL. This wasshaken at room temperature for one hour for complex formation.

Next, solutions of the antibody alone or the antibody as a complex wereadded to the plate from which the blocking solution had been removed,and this was shaken at room temperature for one hour. Then, afterremoval of the antibody-alone solution or the complex solution, Buffer-1containing 0.25% Glutaraldehyde was added. Then, after the plate wasallowed to stand for 10 minutes, it was washed with DPBS (manufacturedby Wako Pure Chemical Industries) containing 0.05% Tween 20. An antibodyfor ECL detection was prepared by sulfo-tagging the goat anti-human IgG(gamma) (manufactured by Zymed Laboratories) using the Sulfo-Tag NHSEster (manufactured by MSD). The antibody for ECL detection was dilutedwith Buffer-2 to be 1 μg/mL, added to the plate, and then shaken in thedark at room temperature for one hour. The antibody for ECL detectionwas removed, a solution produced by 2-fold dilution of the MSD ReadBuffer T (4×) (manufactured by MSD) using ultrapure water was added, andthen the amount of luminescence was measured by SECTOR Imager 2400(manufactured by MSD).

The results are shown in FIG. 30. Interestingly, all of the anti-IL-8antibodies such as H1009/L395 hardly showed any binding to theextracellular matrix as the antibody alone (-IL8), but bound to theextracellular matrix upon complex formation with human IL-8 (+hIL8).

As described above, the property of anti-IL-8 antibodies to acquireaffinity for the extracellular matrix by binding to human IL-8 has notbeen elucidated. Furthermore, without being limited, combining suchproperties with pH-dependent IL-8-binding antibodies can increase therate of IL-8 elimination more efficiently.

Example 16 Mouse PK Assay Using Non-FcRn-Binding Antibodies

The following method was used to confirm whether a complex between humanIL-8 and a pH-dependent IL-8-binding antibody is formed and uptake ofthat complex into cells increases in mice.

First, an antibody variant comprising the variable region of H1009/L395and an Fc region deficient in binding affinity to various Fc receptorswas produced. Specifically, as modifications for deleting the bindingability towards human FcRn under acidic pH conditions, the heavy chainH1009-IgG1 was subjected to substitution of alanine for isoleucine atposition 253 and aspartic acid for serine at position 254, according toEU numbering. Furthermore, as modifications for deleting the binding tomouse FcγR(s), leucine at position 235 was substituted with arginine,glycine at position 236 was substituted with arginine, and serine atposition 239 was substituted with lysine. 1009-F1942m (SEQ ID NO:93) wasproduced as a heavy chain containing four of these modifications.Furthermore, H1009/L395-F1942m comprising H1009-F1942m as the heavychain and L395-k0MT as the light chain was produced.

Since antibody that has this Fc region is deficient in the FcRn bindingaffinity under acidic pH conditions, it is not transferred from theendosome into plasma. Therefore, such antibody is quickly eliminatedfrom plasma in a living body as compared to antibody that comprisesnative Fc region. In this case, after the antibody that comprises nativeFc region is incorporated into cells, only a portion of them that is notsalvaged by FcRn is degraded after being transferred to the lysosome,but in the case of antibody comprising Fc region that does not compriseFcRn-binding affinity, all of the antibody incorporated into the cellsare degraded in lysosomes. More specifically, in the case of antibodythat comprise such modified Fc region, the rate of elimination of theadministered antibody from plasma may be equivalent to the rate ofincorporation into cells. That is, the rate of intracellular uptake ofthe antibody whose FcRn-binding affinity has been deleted can also beconfirmed by measuring the rate of elimination of these antibodies fromplasma.

Then, whether intracellular uptake of the complex formed betweenH1009/L395-F1942m and human IL-8 increases as compared to the uptake ofH1009/L395-F1942m alone was tested. Specifically, whether the rate ofelimination of the antibody from plasma will change when the antibody isadministered alone and when the antibody is administered upon formationof a complex with human IL-8 was tested.

The respective biokinetics of the anti-human IL-8 antibody was evaluatedin cases when the anti-human IL-8 antibody was administered alone tohuman FcRn transgenic mice (B6.mFcRn−/−.hFcRn Tg line 32+/+ mouse;Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)) and whenhuman IL-8 and the anti-human IL-8 antibody were administeredsimultaneously to the human FcRn transgenic mice. The anti-human IL-8antibody solution (200 μg/mL), and a mixed solution of human IL-8 (10μg/mL) and the anti-human IL-8 antibody (200 μg/mL) were individuallyadministered once at 10 mL/kg to the tail vein. In this case, since theanti-human IL-8 antibody was present in sufficient excess over humanIL-8, almost all of human IL-8 was considered to be bound to theantibody. Blood was collected five minutes, two hours, seven hours, oneday, and two days after the administration. The collected blood wasimmediately centrifuged at 4° C. and 15,000 rpm for 15 minutes to obtainplasma. The separated plasma was stored in a freezer set to −20° C. orbelow until measurements were taken.

The anti-human IL-8 antibody concentration in mouse plasma was measuredby an electrochemiluminescence method. First, to the Streptavidin GoldMulti-ARRAY Plate (Meso Scale Discovery) which had been blockedovernight at room temperature using a PBS-Tween solution containing 5%BSA (w/v), an Anti-Human Kappa Light Chain Goat IgG Biotin (IBL) wasallowed to react at room temperature for one hour to produce ananti-human antibody-immobilized plate. Samples for calibration curvecontaining the anti-human IL-8 antibody at concentrations of 3.20, 1.60,0.800, 0.400, 0.200, 0.100, and 0.0500 μg/mL in plasma and samples formouse plasma measurement diluted 100-fold or higher were prepared. Eachsample was mixed with human IL-8, and then dispensed at 50 μL per wellinto the anti-human antibody-immobilized plate, and then stirred at roomtemperature for one hour. Human IL-8 was adjusted to a finalconcentration of 333 ng/mL.

Then, an anti-human IL-8 antibody (prepared in-house) comprising a mouseIgG constant region was added to the plate, and was allowed to react atroom temperature for one hour. Furthermore, the Anti-Mouse IgG (BECKMANCOULTER) ruthenium-labeled with the SULFO-TAG NHS Ester (Meso ScaleDiscovery) was added to the plate, and this was allowed to react for onehour. Then, immediately after the Read Buffer T(×1) (Meso ScaleDiscovery) was dispensed into the plate, measurement was carried outusing SECTOR Imager 2400 (Meso Scale Discovery). The anti-human IL-8antibody concentration was calculated based on the response in thecalibration curve using the analytical software, the SOFTmax PRO(Molecular Devices).

Antibody concentrations in mouse plasma obtained as a result are shownin FIG. 31, and the antibody clearance under the respective conditionsare shown in Table 24.

TABLE 24 IL8 CL Antibody Name μg/kg mL/d/kg H1009/L395-F1942m — 134H1009/L395-F1942m 100 291

The rate of intracellular uptake of the complex of H1009/L395-F1942m andhuman IL-8 was shown to be increased by at least 2.2 fold compared tothe uptake rate of H1009/L395-F1942m. Here, it is noted as “at least2.2-fold” because of the following reason which is included as one ofthe possibilities that the value may actually be 5-fold, 10-fold, or30-fold. As the rate of elimination of human IL-8 from mouse plasma isvery rapid compared to the rate of elimination of H1009/L395-F1942m, theproportion of H1009/L395-F1942m bound by human IL-8 in plasma quicklydecreases after administration. More specifically, even whenadministered simultaneously with human IL-8, not all H1009/L395-F1942mpresent in the plasma are in the human IL-8-bound form, and in fact, atapproximately seven hours after administration, most of them alreadyexist in the free form. Since the uptake rate is evaluated under suchconditions, even if the rate of intracellular uptake of the complex ofH1009/L395-F1942m and human IL-8 has been actually increased five-fold,ten-fold, or 30-fold in comparison to the uptake rate ofH1009/L395-F1942m, the results in this experiment system are reflectedonly partially; therefore, the effect may possibly be presented as anincrease of 2.2-fold or so. Accordingly, from these obtained results,whereas the intracellular uptake rate of the complex of H1009/L395 andIL-8 was shown to be increased compared to the actual intracellularuptake rate of H1009/L395 in vivo, this effect is not limited to theobtained value of 2.2-fold increase.

Without being particularly limited, the following interpretation may bemade from the findings obtained so far. When H1009/L395, which is apH-dependent IL-8-binding antibody, forms a complex with human IL-8,that complex has a higher isoelectric point and is shifted more towardsa positive charge than when the antibody alone exists. At the same time,the affinity of the complex towards the extracellular matrix is moreincreased than the affinity of the antibody alone. Properties such aselevation of isoelectric point and enhancement of the extracellularmatrix binding can be considered as factors that promote uptake of anantibody into cells in vivo. Furthermore, from mouse experiments, therate of intracellular uptake of the complex of H1009/L395 and human IL-8was shown to be increased 2.2-fold or greater compared to the uptakerate of H1009/L395. From the above, the theoretical explanation as wellas the in vitro properties and in vivo phenomena consistently supportthe hypothesis that H1009/L395 and human IL-8 form a complex to promoteuptake of the complex into cells, and leads to a remarkable increase inthe elimination of human IL-8.

Several antibodies against IL-8 have been reported to date, but therehas been no report so far on the increase of binding affinity to theextracellular matrix upon complex formation with IL-8 and the increasein uptake of the complexes into cells.

Furthermore, based on the finding that an increase in the intracellularuptake of the anti-IL-8 antibodies is observed when the antibodies formcomplexes with IL-8, one may consider that the anti-IL-8 antibodies thathave formed complexes with IL-8 in plasma are quickly taken up intocells, while the free antibodies which have not formed complexes withIL-8 tend to be retained in plasma without being taken up into cells. Inthis case, when the anti-IL-8 antibody is pH-dependent, the anti-IL-8antibody which has been taken up into the cells releases the IL-8molecule in the cells and then returns to the outside of the cells, andthen it can bind to another IL-8 molecule; and therefore, increase inthe intracellular uptake upon complex formation may have a furthereffect of eliminating IL-8 more strongly. That is, selecting anti-IL-8antibodies with increased binding to the extracellular matrix oranti-IL-8 antibodies with increased uptake into cells may also beanother embodiment of Disclosure C.

Example 17 Immunogenicity Prediction of the pH-Dependent IL-8-BindingAntibody H1009/L395 Using an in Silico System

Next, the immunogenicity score and frequency of ADA development werepredicted for H1009/L395 by a method similar to that of Example 13-1.The results are shown in Table 25 and FIG. 32. In FIG. 32, H1009/L395 isnoted as “H1009L395”.

TABLE 25 Heavy Chain Light Chain Total Antibody EpiMatrix tReg AdjustedEpiMatrix tReg Adjusted EpiMatrix tReg Adjusted Name Score Epx ScoreScore Epx Score Score Epx Score hWS-4 62.44 12.18 22.64 −23.89 85.08−11.71 H1004/L395 10.79 −39.47 7.79 −38.74 18.58 −78.21 H1009/L395 9.62−40.64 7.79 −38.74 17.41 −79.38

The results in Table 25 show that H1009/L395 has the same level of lowimmunogenicity scores as H10041L395. Furthermore, the frequency of ADAdevelopment predicted for H1009/L395 from the results in FIG. 32 was 0%,and this was also similar to that of H1004/L395.

Accordingly, the predicted immunogenicity was greatly decreased forH1009/L395 in comparison to the known anti-human IL-8 antibody hWS-4.Therefore, H1009/L395 is considered to have very low immunogenicity inhumans, and to be able to stably maintain the anti-IL-8-neutralizingactivity for a long period of time.

Example 18 Cynomolgus Monkey PK Assay Using an H89/L118 Variant withEnhanced FcRn-Binding Ability Under Acidic pH Conditions

As described in the Examples above, among the cases where the antibodieshave native IgG1 as their constant region, the pH-dependent IL-8-bindingantibody H1009/L395 is an antibody that has superior properties.However, such antibodies can also be used as antibodies containing aminoacid substitutions in the constant region, for example, those containingan Fc region with enhanced FcRn binding at acidic pH, as exemplified inExample 5. Therefore, H89/L118 was used to confirm that the Fc regionwith enhanced FcRn binding at acidic pH can also function in apH-dependent IL-8-binding antibody.

(18-1) Production of an H89/L118 Fc Region-Modified Antibody withEnhanced FcRn Binding at Acidic pH

Various modifications for enhancing FcRn binding as described in Example5-1 were introduced into the Fc region of H89/L118. Specifically, thefollowing variants were produced by introducing the modifications usedin F1847m, F1848m, F1886m, F1889m, F1927m, and F1168m into the Fc regionof H89-IgG1: (a) H89/L118-IgG1 comprising H89-IgG1m (SEQ ID NO:94) asthe heavy chain and L118-K0MT as the light chain; (b) H89/L118-F1168mcomprising H89-F1168m (SEQ ID NO:95) as the heavy chain and L118-K0MT asthe light chain; (c) H89/L118-F1847m comprising H89-F1847m (SEQ IDNO:96) as the heavy chain and L118-K0MT as the light chain; (d)H89/L118-F1848m comprising H89-F1848m (SEQ ID NO:97) as the heavy chainand L118-K0MT as the light chain; (e) H89/L118-F1886m comprisingH89-F1886m (SEQ ID NO:98) as the heavy chain and L118-K0MT as the lightchain; (f) H89/L118-F1889m comprising H89-F1889m (SEQ ID NO:99) as theheavy chain and L118-K0MT as the light chain; and (g) H89/L118-F1927mcomprising H89-F1927m (SEQ ID NO:100) as the heavy chain and L118-K0MTas the light chain. Cynomolgus monkey PK assays using these antibodieswere carried out by the method shown below.

(18-2) Cynomolgus Monkey PK Assay of Novel Fc Region Variant-ContainingAntibodies

After administration of anti-human IL-8 antibodies to cynomolgusmonkeys, biokinetics of the anti-human IL-8 antibodies was evaluated. Ananti-human IL-8 antibody solution was intravenously administered once at2 mg/kg. Blood was collected five minutes, four hours, one day, twodays, three days, seven days, ten days, 14 days, 21 days, 28 days, 35days, 42 days, 49 days, and 56 days after administration. The collectedblood was immediately centrifuged at 4° C. and 15,000 rpm for tenminutes to obtain plasma. The separated plasma was stored in a freezerset to −60° C. or below until measurements were taken.

The anti-human IL-8 antibody concentration in cynomolgus monkey plasmawas measured by an electrochemiluminescence method. First, theAnti-hKappa Capture Ab (Antibody Solutions) was dispensed into aMULTI-ARRAY 96-well Plate (Meso Scale Discovery), and was stirred atroom temperature for one hour. Then, a PBS-Tween solution containing 5%BSA (w/v) was used for blocking at room temperature for two hours toprepare an anti-human antibody-immobilized plate. Samples forcalibration curve containing an anti-human IL-8 antibody atconcentrations of 40.0, 13.3, 4.44, 1.48, 0.494, 0.165, and 0.0549 μg/mLin plasma and samples for cynomolgus monkey plasma measurement diluted500-fold or more were prepared, 50 μL of the solutions were dispensedinto each well of the anti-human antibody-immobilized plate, and thesolutions were stirred at room temperature for one hour. Then, theAnti-hKappa Reporter Ab, Biotin conjugate (Antibody Solutions) was addedto the aforementioned plate, and allowed to react at room temperaturefor one hour. After further adding the SULFO-TAG Labeled Streptavidin(Meso Scale Discovery) and allowing to react at room temperature for onehour, the Read Buffer T(×1) (Meso Scale Discovery) was dispensed intothe plate, and measurements were taken immediately using SECTOR Imager2400 (Meso Scale Discovery). The anti-human IL-8 antibody concentrationwas calculated based on the response in the calibration curve using theanalytical software, the SOFTmax PRO (Molecular Devices).

The results obtained for the half-life (t1/2) and clearance (CL) of eachof the antibodies are shown in Table 26, and changes in the antibodyconcentration in cynomolgus monkey plasma are shown in FIG. 33.

TABLE 26 t½ CL Antibody Name day mL/d/kg H89/L118-IgG1 11.9 2.95H89/L118-F1168m 24.1 3.21 H89/L118-F1847m 27.9 2.09 H88/L118-F1848m 25.31.74 H89/L118-F1886m 45.1 1.34 H89/L118-F1889m 39.5 1.75 H89/L118-F1927m30.3 2.13

The above results confirmed that all of the Fc region variants showprolonged retention in plasma in comparison to the antibody that has anative IgG1 Fc region. In particular, H89/L118-F1886m showed the mostdesirable blood kinetics.

Example 19 Fc Region with Lowered Binding Ability Towards FcγRs

The Fc region of a native human IgG1 is known to bind to Fcγreceptor(s)(hereinafter, referred to as FcγR(s)) on various cells of the immunesystem, and exhibit effector functions such as ADCC and ADCP on targetcells.

On the other hand, IL-8 is a soluble cytokine, and anti-IL-8 antibodiesused as pharmaceuticals are mainly expected to show pharmacologicalactions by neutralizing the functions of IL-8 at sites where IL-8 ispresent in excess. Such sites where IL-8 is present in excess are notparticularly limited, and for example, may be inflamed sites. It isknown that generally at such inflamed sites, various immune cells gatherand are activated. Transmitting unintended activation signals to thesecells via Fc receptors and inducing activities such as ADCC and ADCP inunintended cells are not always favorable. Therefore, without beingparticularly limited, from a safety point of view, it may be preferablethat anti-IL-8 antibodies administered in vivo have low affinity towardsFcγRs.

(19-1) Production of Modified Antibodies with Lowered Binding TowardsFcγRs

Amino acid modifications were further introduced into the Fc region ofH1009/L395-F1886m with the objective of reducing the binding abilitytowards various human and cynomolgus monkey FcγRs. Specifically,H1009-F1886s (SEQ ID NO:81) was produced by subjecting the H1009-F1886mheavy chain to each of the following substitutions: R for L at position235, R for G at position 236, and K for S at position 239, according toEU numbering. Similarly, H1009-F1974m (SEQ ID NO:80) was produced bysubjecting H1009-F1886m to substitution of R for L at position 235 and Rfor G at position 236, according to EU numbering, and substituting theregion from position 327 to position 331 according to EU numbering withthat of the native human IgG4 sequence. H1009/L395-F1886s andH1009/L395-F1974m were produced as antibodies having these heavy chains,and L395-k0MT as the light chain.

(19-2) Confirmation of the Affinity Towards Various Human FcγRs

Next, the affinities of the H1009/L395-F1886s or H1009/L395-F1974mtowards the soluble forms of FcγRIa or FcγRIIIa in human or cynomolgusmonkey were confirmed by the following method.

Assays were performed for the binding of the H1009/L395-F1886s or theH1009/L395-F1974m to the soluble forms of FcγRIa or FcγRIIIa in human orcynomolgus monkey using BIACORE T200 (GE Healthcare). Soluble FcγRIa andFcγRIIIa in both human and cynomolgus monkey were produced in the formof His-tagged molecules by methods known to those of ordinary skill inthe art. An appropriate amount of rProtein L (BioVision) was immobilizedonto the Sensor chip CM4 (GE Healthcare) by the amine coupling methodand antibody of interest was captured. Next, soluble FcγRIa or FcγRIIIawas injected with a running buffer (used as a reference solution), andwas made to interact with the antibodies captured onto the sensor chip.HBS-EP+ (GE Healthcare) was used as the running buffer, and HBS-EP+ wasalso used to dilute the soluble FcγRIa or FcγRIIIa. To regenerate thesensor chip, 10 mM glycine-HCl at pH 1.5 was used. All measurements werecarried out at 20° C.

The results are shown in FIG. 34. Here, the notations used for humanFcγRIa, human FcγRIIIa, cynomolgus monkey FcγRIa, and cynomolgus monkeyFcγRIIIa are in the same order: hFcγRIa, hFcγRIIIa, cynoFcγRIa, andcynoFcγRIIIa, respectively. H1009/L395-F1886m was shown to bind to allFcγRs, but on the other hand, the H1009/L395-F1886s andH1009/L395-F1974m were confirmed not to bind to any of the FcγRs.

(19-3) Mouse IL-8 Elimination Assay of Fc Variants

Next, for the H1009/L395-F1886s and H1009/L395-F1974m, the rate of humanIL-8 elimination and the retention in plasma of the antibodies in micewere confirmed by the following experiment. Here, three doses ofH1009/L395-F1886s, 2 mg/kg, 5 mg/kg, and 10 mg/kg, were used for theevaluation so that the effects of increasing the antibody dosage canalso be evaluated for H1009/L395-F1886s.

After simultaneous administration of human IL-8 and an anti-human IL-8antibody to human FcRn transgenic mice (B6.mFcRn−/−.hFcRn Tg line 32+/+mouse; Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)), thebiokinetics of human IL-8 was evaluated. A mixed solution of human IL-8(10 μg/mL) and an anti-human IL-8 antibody (200 μg/mL, 500 μg/mL, or1000 μg/mL) was administered once at 10 mL/kg through the tail vein. Inthis case, since the anti-human IL-8 antibody was present in sufficientexcess over human IL-8, almost all of human IL-8 was considered to bebound to the antibody. Blood was collected five minutes, two hours, fourhours, seven hours, one day, two days, three days, seven days, 14 days,21 days, and 28 days after the administration. The collected blood wasimmediately centrifuged at 4° C. and 15,000 rpm for 15 minutes to obtainplasma. The separated plasma was stored in a freezer set to −20° C. orbelow until measurements were taken.

The human IL-8 concentration in mouse plasma was measured by a methodsimilar to that of Example 11. The resulting data on the human IL-8concentration in plasma is shown in FIG. 35, and the values of humanIL-8 clearance (CL) from mouse plasma are shown in Table 27.

First, H1009/L395 comprising the Fc region of a native IgG1 andH1009/L395-F1886s comprising the modified Fc region were shown to haveequivalent human IL-8-eliminating effects when the 2 mg/kg-administeredgroups were compared.

Next, when the dosage of the H1009/L395-F1886s antibody was changed,significant difference in the human IL-8 clearance values was notobserved between the 2 mg/kg and 10 mg/kg doses while there was a slightdifference in the plasma IL-8 concentration one day afteradministration. This strongly suggests that antibodies comprising thevariable region of H1009/L395 showed sufficient IL-8-eliminating effectseven when the antibodies were administered at high doses.

TABLE 27 Human IL-8 CL Antibody Name Dose (mL/d/kg) H1009/L395 2 mg/kg740 H1009/L395-F1886s 2 mg/kg 628 H1009/L395-F1886s 5 mg/kg 458H1009/L395-F1886s 10 mg/kg 560(19-4) Cynomolgus Monkey PK Assay of Fc Variants

Next, plasma retention of antibodies in cynomolgus monkeys was verifiedby the following method using H1009/L395-F1886s or H1009/L395-F1974m.

Biokinetics of an anti-human IL-8 antibody were evaluated in case thatthe anti-human IL-8 antibody was administered alone or in case thathuman IL-8 and the anti-human IL-8 antibody were simultaneouslyadministered to cynomolgus monkeys. An anti-human IL-8 antibody solution(2 mg/mL) or a mixed solution of human IL-8 (100 μg/kg) and ananti-human IL-8 antibody (2 mg/kg) was intravenously administered onceat 1 mL/kg. Blood was collected five minutes, four hours, one day, twodays, three days, seven days, ten days, 14 days, 21 days, 28 days, 35days, 42 days, 49 days, and 56 days after administration. The collectedblood was immediately centrifuged at 4° C. and 15,000 rpm for tenminutes to obtain plasma. The separated plasma was stored in a freezerset to −60° C. or below until measurements were taken.

The anti-human IL-8 antibody concentration in cynomolgus monkey plasmawas measured by the method of Example 18. The resulting data on theanti-human IL-8 antibody concentration in plasma is shown in FIG. 36,and the values for the half-life (t_(1/2)) and clearance (CL) of theanti-human IL-8 antibody from cynomolgus monkey plasma are shown inTable 28.

First, in comparison to Hr9 and H89/L118 which have the Fc region of anative human IgG1, H1009/L395-F1886s which has an Fc region withimproved functions was shown to have significantly prolonged plasmaretention.

Furthermore, when H1009/L395-F1886s was administered simultaneously withhuman IL-8, the change in plasma concentration was equivalent to thatwhen the antibody was administered alone. Without being particularlylimited, the following discussion is possible from this finding. Asdescribed above, intracellular uptake of the complex of H1009/L395 andhuman IL-8 has been shown to be increased compared to the uptake ofH1009/L395 alone. Generally, high-molecular-weight proteins are thoughtto be incorporated non-specifically or in a receptor-dependent mannerinto cells, then transferred to the lysosome and degraded by variousdegrading enzymes present in the lysosome. Therefore, if the rate ofuptake of the protein into cells increases, the plasma retention of thatprotein is likely to worsen as well. However, in the case of anantibody, it has the property of being returned to the plasma by FcRn inthe endosome; and therefore, as long as the salvaging by FcRn functionssufficiently, plasma retention may not be affected even if the rate ofintracellular uptake is accelerated. Here, even when H1009/L395-F1886swas administered simultaneously with human IL-8 to cynomolgus monkeys,plasma retention was not affected. This indicates the possibility thatwhile the rate of antibody uptake into cells is increased forH1009/L395-F1886s, the antibody is sufficiently salvaged by FcRn suchthat it can return to the plasma.

Furthermore, another Fc variant H1009/L395-F1974m also showed equivalentplasma retention to that of H1009/L395-F1886s. While these Fc variantshave been introduced with different modifications that decrease thebinding ability to various FcγRs as describe above, they have been shownnot to affect the plasma retention of the antibodies themselves. Fromthe above, plasma retention of both H1009/L395-F1886s andH1009/L395-F1974m in cynomolgus monkeys was shown to be remarkablyprolonged and extremely satisfactory in comparison to that of antibodiesthat have the native IgG1 Fc region.

TABLE 28 t½ CL day mL/d/kg Hr9 20.26 3.72 H89/L118 11.88 2.95H1009/L395-F1886s 35.75 1.64 H1009/L395-F1886s + hIL-8 72.24 1.11H1009/L395-F1974m + hIL-8 43.78 1.60

As demonstrated in the above-mentioned Examples, by comprisingpH-dependent IL-8 binding ability with a feature of being quickly takenup into cells as a complex with IL-8, H1009/L395 achieved for the firsttime as an antibody that increases significantly the rate of human IL-8elimination in vivo. Furthermore, the IL-8-binding affinity of thisantibody under neutral pH conditions is also increased compared to theknown hWS-4 antibody, and the antibody can neutralize human IL-8 morestrongly under neutral pH conditions such as in plasma. In addition, itis an antibody that has excellent stability under plasma conditions, andwhose IL-8 neutralizing activity does not decrease after it isadministered in vivo. Furthermore, H1009/L395, constructed based on Hr9which has a greatly improved production level as compared to the hWS-4,is an antibody suitable for manufacturing from the viewpoint ofproduction level. Moreover, in in silico immunogenicity prediction, theantibody showed a very low score for its immunogenicity, and this scorewas significantly lower in comparison to those of the known hWS-4antibody and several other known commercially available antibodies. Thatis, it is expected that H1009/L395 would hardly generate ADA in humans,and would be able to be used safely for a long period of time.Accordingly, in comparison to known anti-human IL-8 antibodies,H1009/L395 shows improvement in various aspects, and is very useful as apharmaceutical.

H1009/L395 which has the native IgG Fc region is sufficiently useful asdescribed above; however, variants of H1009/L395 comprising thefunctionally-improved Fc region can also be used appropriately asantibodies with enhanced utility. Specifically, it is possible toincrease the FcRn binding under acidic pH conditions to prolong plasmaretention and to maintain effects for a longer period of time.Furthermore, variants comprising the Fc region introduced withmodification(s) that decrease the binding ability to FcγR(s) can be usedas high-safety therapeutic antibodies to avoid unintended activation ofimmune cells and generation of cytotoxic activity in the administeredorganism. As such Fc variants, the use of F1886s or F1974m exploitedherein is particularly favorable, but it is not limited to these Fcvariants; and as long as the Fc variant has similar functions,therapeutic antibodies comprising other modified Fc regions are used asan embodiment of Disclosure C.

As a result, the antibodies of Disclosure C including H1009/L395-F1886sand H1009/L395-F1974m generated by the inventors through dedicatedresearch can maintain a condition where the biological activity of humanIL-8 is strongly inhibited both safely and for a long period of time.Here, levels that could not be achieved by known anti-IL-8 antibodieshave been realized, and these antibodies of Disclosure C are expected tobe used as high-quality finished anti-IL-8 antibody pharmaceuticals.

Example 20 Anti-Factor IXa/Factor X Bispecific Antibodies

The humanized anti-factor IXa/factor X bispecific antibodies disclosedin WO2012/067176 bind to human factor IXa and factor X and induceco-aggregation activity of blood. A humanized anti-factor IXa/factor Xbispecific antibody F8M(Q499-z121/J327-z119/L404-k:H chain (SEQ IDNO:330)/H chain (SEQ ID NO:331)/common L chain (SEQ ID NO:332))described in WO2012/067176 was utilized in this example and F8Mcomprises two different H chains and two same common L chains. F8M wasproduced by the method described in Examples of WO2012/067176.

(20-1) Production of Anti-Factor IXa/Factor X Bispecific Antibodies

The following three antibodies were produced by the method of ReferenceExample 2 as anti-factor IXa/factor X bispecific antibodies based onF8M: (a) F8M-F1847mv, which is a conventional antibody comprisingF8M-F1847mv1 (SEQ ID NO:323) and F8M-F1847mv2 (SEQ ID NO:324) as theheavy chains and F8ML (SEQ ID NO:325) as the light chain; (b)F8M-F1868mv, which is a conventional antibody comprising F8M-F1868mv1(SEQ ID NO:326) and F8M-F1868mv2 (SEQ ID NO:327) as the heavy chains andF8ML (SEQ ID NO:325) as the light chain; and (c) F8M-F1927mv, which is aconventional antibody comprising F8M-F1927mv1 (SEQ ID NO:328) andF8M-F1927mv2 (SEQ ID NO:329) as the heavy chains and F8ML (SEQ IDNO:325) as the light chain.

The heavy chain sequences include the same Fc variant sequencesregarding the enhancement of FcRn binding and the reduction of therheumatoid factor binding mentioned in Example 5 as follows:

TABLE 29 Sequence Name Name in Example 5 F8M-F1847mv1 (SEQ ID NO: 323)F1847m F8M-F1847mv2 (SEQ ID NO: 324) F1847m F8M-F1868mv1 (SEQ ID NO:326) F1868m F8M-F1868mv2 (SEQ ID NO: 327) F1868m F8M-F1927mv1 (SEQ IDNO: 328) F1927m F8M-F1927mv2 (SEQ ID NO: 329) F1927m(20-2) Pharmacokinetic Study of Monoclonal Antibodies, F8M-F1847mv,F8M-F1868mv, and F8M-F1927mv, in Cynomolgus Monkey

Pharmacokinetics of monoclonal antibodies, F8M-F1847mv, F8M-F1868mv, andF8M-F1927mv, after single bolus intravenous administration at the doseof 0.6 mg/kg to male cynomolgus monkey were each evaluated. The plasmaconcentrations of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv weredetermined by a sandwich ELISA. The pharmacokinetic parameters werecalculated using WinNonlin ver 6.4 software. As shown in Table 30, thehalf-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were 29.3 day,54.5 day, and 35.0 day, respectively. The PK study of F8M usingcynomolgus monkey was conducted in a different day at the dose of 6mg/kg, and the half-life was revealed to be 19.4 day. It was clarifiedthat the half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv werelonger than F8M. This suggests that the half-life of an anti-factorIXa/X bispecific antibody could be prolonged by the same modification onthe Fc region sequence with that mentioned in Example 5 above.

TABLE 30 Half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv and F8Mafter intravenous administration to male cynomolgus monkey F8M-F1847mvF8M-F1868mv F8M-F1927mv F8M Half-life (day) 29.3 54.5 35.0 19.4

Example 21 Evaluation of Clearance of IgE from Plasma Using pI-IncreasedFab Variants

To enhance the clearance of human IgE, pI increased substitutions in theFab portion of antibodies were evaluated in this example usingpH-dependent antigen-binding antibodies. The method of adding amino acidsubstitutions to the antibody variable region to increase pI is notparticularly limited, but for example, it can be performed by the methoddescribed in WO2007/114319 or WO2009/041643. Amino acid substitutionsintroduced into the variable region are preferably those that decreasethe number of negatively charged amino acids (such as aspartic acid andglutamic acid) while increasing the positively charged amino acids (suchas arginine and lysine). Furthermore, amino acid substitutions may beintroduced at any position in the antibody variable region. Withoutparticular limitation, the sites for introducing amino acidsubstitutions are preferably positions where amino acid side chains maybe exposed on the antibody molecule surface.

(21-1) Production of Antibodies with Increased pI by Modification ofAmino Acids in the Variable Region

The tested antibodies are summarized in Table 32 and Table 33.

The heavy chain, Ab1H003 (also called H003, SEQ ID NO:144) was preparedby introducing pI-increasing substitution H32R into Ab1H (SEQ ID NO:38).Other heavy chain variants were also prepared by introducing respectivesubstitutions represented in Table 32 into Ab1H according to the methodshown in Reference Example 1. All the heavy chain variants wereexpressed with Ab1L (SEQ ID NO:39) as light chain. The pH-dependentbinding profile of this antibody is summarized in Table 5 (Ab1).

Similarly, we also evaluated the pI-increasing substitution in lightchain.

The light chain, Ab1L001T (also called L001, SEQ ID NO:164) was preparedby introducing pI-increasing substitution G16K into Ab1L. Other lightchain variants were also prepared by introducing respectivesubstitutions represented in Table 33 into Ab1L according to the methodshown in Reference Example 1. All the light chain variants wereexpressed with Ab1H as heavy chain.

TABLE 32 Heavy Chain Variants of Ab1H evaluated in this Example SampleName (H Chain/ Imaging BIACORE L Chain) Variant fold fold Ab1H/Ab1LOriginal 1.00 1.00 Ab1 Ab1H003/Ab1L H003 H32R no data no dataAb1H005m/Ab1L H005 P41R/G44R 1.73 0.92 Ab1H010/Ab1L H010 T77R 1.68 1.05Ab1H012/Ab1L H012 D82aN/S82bR 2.71 0.96 Ab1H013/Ab1L H013 D82aG/S82bR2.95 1.02 Ab1H014/Ab1L H014 D82aS/S82bR 2.36 0.91 Ab1H016/Ab1L H016 E85G1.51 1.04 Ab1H018/Ab1L H018 A93K 0.00 −0.01   Ab1H026m/Ab1L H026P41R/G44R/T77R 0.76 no data Ab1H027/Ab1L H027 T77R/D82aN/S82bR 2.78 1.21Ab1H028/Ab1L H028 T77R/D82aG/S82bR 3.04 1.37 Ab1H029/Ab1L H029T77R/D82aS/S82bR 1.80 1.33 Ab1H030/Ab1L H030 T77R/E85G 1.50 1.27Ab1H031m/Ab1L H031 T77R/A93K 0.03 0.06 Ab1H032/Ab1L H032D82aG/S82bR/E85G 0.52 no data Ab1H034/Ab1L H034 Q13K 1.12 1.21Ab1H035/Ab1L H035 G15R 0.06 1.28 Ab1H039/Ab1L H039 S64K 0.77 1.57Ab1H041m/Ab1L H041 Q105R 1.03 1.52 Ab1H045/Ab1L H045 S82bR 2.00 0.76

TABLE 33 Light Chain Variants of Ab1L evaluated in this Example SampleName (H Chain/ Imaging BIACORE L Chain) Variant fold fold Ab1H/Ab1LOriginal 1.00 1.00 Ab1 Ab1H/Ab1L001 L001 G16K 2.11 1.03 Ab1H/Ab1L002L002 Q24R/E27Q 1.43 1.03 Ab1H/Ab1L003 L003 Q24R/E27R 3.14 1.09Ab1H/Ab1L004 L004 Q24K/E27K 1.42 1.01 Ab1H/Ab1L005 L005 A25K/S26K 1.820.84 Ab1H/Ab1L006 L006 A25R/S26R 6.82 1.18 Ab1H/Ab1L007 L007 Q37R 1.821.06 Ab1H/Ab1L008 L008 G41R/Q42K 1.70 1.07 Ab1H/Ab1L009 L009 L46R/Y49K0.02 −0.02   Ab1H/Ab1L010 L010 S52R/S56R 2.72 0.98 Ab1H/Ab1L011 L011S52K/S56K 1.21 1.02 Ab1H/Ab1L012 L012 S65R/T69R 1.28 0.97 Ab1H/Ab1L013L013 T74K/S77R 3.31 1.70 Ab1H/Ab1L014 L014 S76R/Q79K 4.47 1.08Ab1H/Ab1L015 L015 G16K/Q24R/E27R 2.11 1.25 Ab1H/Ab1L016 L016Q24R/E27R/Q37R 3.33 1.36 Ab1H/Ab1L017 L017 Q24R/E27R/G41R/Q42K 2.90 1.27Ab1H/Ab1L018 L018 Q24R/E27R/L46R/Y49K 0.01 0.07 Ab1H/Ab1L019 L019Q24R/E27R/S52R/S56R 3.88 1.17 Ab1H/Ab1L020 L020 Q24R/E27R/S52K/S56K 4.611.22 Ab1H/Ab1L021 L021 Q24R/E27R/S65R/T69R 11.43 1.36 Ab1H/Ab1L022 L022Q24R/E27R/T74K/S77R 19.05 1.45 Ab1H/Ab1L023 L023 Q24R/E27R/S76R/Q79K13.15 1.39 Ab1H/Ab1L024 L024 G16K/A25R/S26R 0.73 No data Ab1H/Ab1L025L025 A25R/S26R/Q37R 2.03 1.39 Ab1H/Ab1L026 L026 A25R/S26R/G41R/Q42K 1.28No data Ab1H/Ab1L028 L028 A25R/S26R/S52R/S56R 6.33 1.46 Ab1H/Ab1L029L029 A25R/S26R/S52K/S56K 9.84 1.23 Ab1H/Ab1L030 L030 A25R/S26R/S65R/T69R7.19 1.16 Ab1H/Ab1L032 L032 A25R/S26R/S76R/Q79K 2.67 No dataAb1H/Ab1L033 L033 Q24R/E27R/G41R/Q42K/S65R/T69R 6.68 1.26 Ab1H/Ab1L034L034 Q24R/E27R/S52R/S56R/S65R/T69R 9.81 1.71 Ab1H/Ab1L035 L035Q24R/E27R/S65R/T69R/T74K/S77R 19.56 1.49 Ab1H/Ab1L036 L036Q24R/E27R/S65R/T69R/S76R/Q79K 17.04 1.48 Ab1H/Ab1L037 L037Q24R/E27R/G41R/Q42K/T74K/S77R 8.62 1.38 Ab1H/Ab1L038 L038Q24R/E27R/S52R/S56R/T74K/S77R 15.13 1.47 Ab1H/Ab1L039 L039Q24R/E27R/T74K/S76R/S77R/Q79K 26.95 0.99 Ab1H/Ab1L040 L040Q24R/E27R/G41R/Q42K/S76R/Q79K 5.29 1.23 Ab1H/Ab1L041 L041Q24R/E27R/S52R/S56R/S76R/Q79K 11.86 1.35 Ab1H/Ab1L061 L061 Q42K/S76R4.86 1.02 Ab1H/Ab1L062 L062 S65R/Q79K 2.93 0.98(21-2) Human FcγRIIb-Binding Assay by BIACORE Using pI-IncreasedVariants

Regarding the produced Fc region variant-containing antibodies, bindingassays between soluble human FcγRIIb and antigen-antibody complexes wereperformed using BIACORE T200 (GE Healthcare). Soluble human FcγRIIb(NCBI accession NM_004001.3) was produced in the form of a His-taggedmolecule by a method known in the art. An appropriate amount of ananti-His antibody was fixed onto Sensor chip CM5 (GE Healthcare) by theamine coupling method using a His capture kit (GE Healthcare) to capturehuman FcγRIIb. Next, an antibody-antigen complex and a running buffer(as a reference solution) were injected, and interaction was allowed totake place with the human FcγRIIb captured onto the sensor chip. 20 mMN-(2-Acetamido)-2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl₂,and 0.05% (w/v) Tween 20 at pH 7.4 was used as the running buffer, andthe respective buffer was also used to dilute the soluble human FcγRIIb.To regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. Allmeasurements were carried out at 25° C. Analyses were performed based onbinding (RU) calculated from sensorgrams obtained by the measurements,and relative values when the binding amount of Ab1H/Ab1L (original Ab1)was defined as 1.00 are shown. To calculate the parameters, the BIACORET100 Evaluation Software (GE Healthcare) was used.

The SPR analysis results are summarized in Tables 32 and 33. A fewvariants were shown to have enhanced binding toward human FcγRIIb fixedon the BIACORE sensor chip.

The antibodies produced by introducing the pI-increasing modification(s)into the variable region are antibodies in which the charge of thevariable region is more positively charged when compared with thosebefore introduction of the modification(s). Therefore, the Coulombicinteraction between the variable region (positive charge) and the sensorchip surface (negative charge) can be considered to have beenstrengthened by the pI-increasing amino acid modifications. Furthermore,such effects are expected to take place similarly on the same negativelycharged cell membrane surface; therefore, they are also expected to showan effect of accelerating the speed of uptake into cells in vivo.

Here, about 1.2 fold or more of the binding to hFcγRIIb of the variantscompared to the binding to hFcγRIIb of original Ab1 was considered tohave strong charge effect on binding of an antibody to hFcγRIIb on thesensor chip.

Among the pI-increased heavy chain variants, the antibody with Q13K,G15R, S64K, T77R, D82aN, D82aG, D82aS, S82bR, E85G or Q105Rsubstitution(s) (according to Kabat numbering), alone or in combination,showed higher binding to hFcγRIIb. The single amino acid substitution orcombination of these substitutions in heavy chain is supposed to havestrong charge effect on binding to hFcγRIIb on the sensor chip. Thus,one or more of positions that are expected to show an effect ofaccelerating the speed or rate of uptake into cells in vivo byintroducing the pI-increasing modification into the heavy chain variableregion of an antibody can include, for example, positions 13, 15, 64,77, 82a, 82b, 85 and 105 according to Kabat numbering. An amino acidsubstitution introduced at such position(s) can be asparagine, glycine,serine, arginine or lysine, and preferably arginine or lysine.

In pI-increased light chain variants, the antibody with G16K, Q24R,A25R, S26R, E27R, Q37R, G41R, Q42K, S52K, S52R, S56K, S56R, S65R, T69R,T74K, S76R, S77R, Q79K substitution(s) (according to Kabat numbering),alone or in combination, shows higher binding to human FcγRIIb. Thesingle amino acid substitution or combination of these substitutions inlight chain is supposed to have strong charge effect on binding to humanFcγRIIb on the sensor chip. Thus, one or more of positions that areexpected to show an effect of accelerating the speed or rate of uptakeinto cells in vivo by introducing the pI-increasing modification intothe light chain variable region of an antibody can include, for example,positions 16, 24, 25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77,and 79 according to Kabat numbering. An amino acid substitutionintroduced at such position(s) can be arginine or lysine.

(21-3) Cellular Uptake of pI-Increased Fab Region Variant-ContainingAntibodies

To evaluate the rate of intracellular uptake into an hFcγRIIb-expressingcell line using the produced Fab region variant-containing antibodies,the assay similar to (4-5) above was performed, provided that the amountof antigen taken up was presented as relative values to the Ab1H/Ab1L(original Ab1) value which is taken as 1.00.

The quantification results of cellular uptake were summarized in Tables32 and 33. Strong fluorescence derived from the antigen in the cells wasobserved in several Fc variants. Here, about 1.5 fold or more of thefluorescence intensity of the antigen taken up into the cells of thevariants compared to the fluorescence intensity of original Ab1 wasconsidered to have strong charge effect on an antigen taken up into thecells.

Among the pI-increased heavy chain variants, the antibody with P41R,G44R, T77R, D82aN, D82aG, D82aS, S82bR or E85G substitution(s)(according to Kabat numbering), alone or in combination, showed strongerantigen uptake into the cells. The single amino acid substitution orcombination of these substitutions in heavy chain is supposed to havestrong charge effect on antigen antibody complex uptake into the cells.Thus, one or more of positions that are expected to cause uptake of anantigen-antibody complex into cells more quickly or more frequently byintroducing the pI-increasing modification into the heavy chain variableregion of an antibody can include, for example, positions 41, 44, 77,82a, 82b or 85, according to Kabat numbering. An amino acid substitutionintroduced at such position(s) can be asparagine, glycine, serine,arginine or lysine, and preferably arginine or lysine.

In pI-increased light chain variants, the antibody with G16K, Q24R,A25R, A25K, S26R, S26K, E27R, E27Q, E27K, Q37R, G41R, Q42K, S52K, S52R,S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitution(s) (according toKabat numbering), alone or in combination, showed stronger antigenuptake into the cells. The single amino acid substitution or acombination of these substitutions in light chain is supposed to havestrong charge effect on antigen antibody complex uptake into the cells.The variants with four or more amino acid substitutions tended to showstronger charge effect than those variants with lesser amino acidsubstitutions. One or more of positions that are expected to causeuptake of an antigen-antibody complex into cells more quickly or morefrequently by introducing the pI-increasing modification into the lightchain variable region of an antibody can include, for example, positions16, 24, 25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77 or 79,according to Kabat numbering. An amino acid substitution introduced atsuch position(s) can be glutamine, arginine or lysine, and preferablyarginine or lysine.

While not being restricted to a particular theory, this result can beexplained as follows: the antigen and antibodies added to the cellculture solution form antigen-antibody complexes in the culturesolution. The antigen-antibody complexes bind to human FcγRIIb expressedon the cell membrane via the antibody Fc region, and are taken up intothe cells in a receptor-dependent manner. Antibodies used in thisexperiment binds to antigen in a pH-dependent manner; therefore, theantibody can dissociate from the antigen in the endosomes (acidic pHconditions) inside the cells. Since the dissociated antigen istransported to lysosome and accumulate, it fluoresces inside the cells.Thus, a strong fluorescence intensity inside the cell is thought toindicate that the uptake of the antigen-antibody complexes into thecells is taking place more quickly or more frequently.

(21-4) Evaluation of Clearance of Human IgE in Mouse Co-Injection Model

Some anti-IgE antibodies with pH-dependent antigen-binding (originalAb1, Ab1H/Ab1L013, Ab1H/Ab1L014, Ab1H/Ab1L007) were tested in miceco-injection model to evaluate their ability to accelerate the clearanceof IgE from plasma. In co-injection model, C57BL6J mice (JacksonLaboratories) were administered by single i.v. injection with IgEpre-mixed with the anti-IgE antibody, respectively. All groups received0.2 mg/kg IgE with 1.0 mg/kg of anti-IgE antibodies. Total IgE plasmaconcentration was determined by anti-IgE ELISA. First, anti-human IgE(clone 107, MABTECH) was dispensed into a microWell plate (Nalge nuncInternational), and left for two hours at room temperature or overnightat 4° C. to prepare an anti-human IgE antibody-immobilized plate.Samples for standard curve and samples were mixed with excess amount ofthe anti-IgE antibody (prepared in house) to form a uniform structure ofimmune complex. These samples were added into the anti-human IgEantibody-immobilized plate, and left for overnight at 4° C. Then, thesesamples were reacted with human GPC3 core protein (prepared in house),biotinized anti-GPC3 antibody (prepared in house), Streptavidin PolyHRP80 Conjugate (Stereospecific Detection Technologies) for one hour inorder. After that, SuperSignal® ELISA Pico Chemiluminescent Substrate(Thermo Fisher Scientific) were added. Chemical luminescence was readwith SpectraMax M2 (Molecular Devices). The concentration of human IgEwas calculated using SOFTmax PRO (Molecular Devices). FIG. 37 describesthe IgE plasma concentration time profile in C57BL6J mice.

After administration of pI-increased Fab variants with pH-dependentantigen-binding, plasma total IgE concentration was lower than that oforiginal Ab1. These results indicate that the antigen-antibody immunecomplex of high pI variants with pH-dependent antigen-binding could bindmore strongly to plasma membrane receptor such as FcγRs, which increasethe cellular uptake of antigen-antibody immune complex. The antigenuptaken into the cells could release from antibody inside the endosomeeffectively, resulted in accelerated elimination of IgE. IgEconcentration of mice treated with Ab1H/Ab1L007, which showed weakefficacy in vitro study, was higher than that of other pI-increase Fabvariant-containing antibodies. These results also suggest that forspeculating an evaluation of clearance of an antigen from plasma invivo, the sensitivity of the in vitro system using the fluorescenceintensity by InCell Analyzer 6000 described above may be higher thanthat of the in vitro BIACORE system described above.

Example 22 Evaluation of Clearance of C5 from Plasma Using pI-IncreasedFab Variants

To enhance the clearance of human IgE, pI-increased substitutions in theFab portion of an antibody were evaluated in this Example usingpH-dependent antigen-binding antibodies.

(22-1) Preparation of C5 [Expression and Purification of RecombinantHuman C5]

Recombinant human C5 (NCBI GenBank accession number: NP 001726.2, SEQ IDNO:207) was expressed transiently using FreeStyle293-F cell line (ThermoFisher, Carlsbad, Calif., USA). Conditioned media expressing human C5was diluted with equal volume of milliQ water, then applied to aQ-sepharose FF or Q-sepharose HP anion exchange column (GE healthcare,Uppsala, Sweden), followed by elution with NaCl gradient. Fractionscontaining human C5 were pooled, then salt concentration and pH wasadjusted to 80 mM NaCl and pH6.4, respectively. The resulting sample wasapplied to a SP-sepharose HP cation exchange column (GE healthcare,Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containinghuman C5 were pooled and subjected to CHT ceramic Hydroxyapatite column(Bio-Rad Laboratories, Hercules, Calif., USA). Human C5 eluate was thenapplied to a Superdex 200 gel filtration column (GE healthcare, Uppsala,Sweden). Fractions containing human C5 was pooled and stored at −150° C.Either in-house prepared recombinant human C5 or plasma derived human C5(CALBIOCHEM, Cat#204888) was used for the study.

Expression and purification of recombinant cynomolgus monkey C5 (NCBIGenBank accession number: XP_005580972, SEQ ID NO:208) was done exactlythe same way as the human counterpart.

(22-2) Preparation of Synthetic Calcium Library

A gene library of antibody heavy chain variable regions which were usedas synthetic human heavy chain libraries consist of 10 heavy chainlibraries. Germ-line frameworks VH1-2, VH1-69, VH3-23, VH3-66, VH3-72,VH4-59, VH4-61, VH4-b, VH5-51, and VH6-1 were selected for this librarybased on germ-line frequency in human B-cell repertoires, andbiophysical properties of V-gene families. The synthetic human heavychain library was diversified at the antibody-binding site mimickinghuman B cell antibody repertoires.

A gene library of antibody light chain variable regions were designed tohave calcium binding motif and were diversified at the positions whichwould contribute to antigen recognition, referring to human B cellantibody repertoires. The design of a gene library of antibody lightchain variable regions which exert characteristics for calcium-dependentbinding to antigens is described in WO 2012/073992.

The combination of a heavy chain variable region library and a lightchain variable region library is inserted in a phagemid vector, and aphage library was constructed, referring to (de Heard et al., Meth. Mol.Biol. 178:87-100 (2002)). A trypsin-cleavage site was introduced intothe phagemid vector at a linker region between Fab and pIII protein.Modified M13K07 helper phage which has a trypsin-cleavage site betweenN2 and CT domains at geneIII was used for Fab displayed phagepreparation.

(22-3) Isolation of Calcium Dependent Anti-C5 Antibodies

The phage display library was diluted with TBS supplemented with BSA andCaCl₂ at the final concentration of 4% and 1.2 mM, respectively. As apanning method, conventional magnetic beads selection was appliedreferring to general protocols (Junutula et al., J. Immunol. Methods332(1-2):41-52 (2008), D'Mello et al., J. Immunol. Methods 247(1-2):191-203 (2001), Yeung et al., Biotechnol. Prog. 18(2):212-220(2002), Jensen et al., Mol. Cell Proteomics 2(2):61-69 (2003). Asmagnetic beads, NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280Streptavidin) were applied. Human C5 (CALBIOCHEM, Cat#204888) waslabelled with EZ-Link NHS-PEG4-Biotin (PIERCE, Cat No. 21329).

The initial round of phage selection, the phage display library wasincubated with biotinylated human C5 (312.5 nM) for 60 minutes at roomtemperature. Phages that displayed binding Fab variants were thencaptured using magnetic beads.

After incubation with beads for 15 minutes at room temperature, thebeads were washed three times with 1 mL of TBS containing 1.2 mM CaCl₂and 0.1% Tween 20, and the beads were washed twice with 1 mL of TBScontaining 1.2 mM CaCl₂. Phages were eluted by re-suspending the beadswith TBS containing 1 mg/mL trypsin for 15 minutes. The eluted phageswere infected with ER2738 and rescued by the helper phage. The rescuedphages were precipitated with polyethylene glycol, re-suspended with TBSsupplemented with BSA and CaCl₂ at the final concentration of 4% and 1.2mM, respectively and used in the next round of panning.

After 1st round of panning, the phages were selected for its calciumdependency, in which the antibody binds to C5 stronger in the presenceof calcium ion. In the second and third round, the panning was performedin the same manner as the first round except by using 50 nM (secondround) or 12.5 nM (third round) of biotinylated antigen and finallyeluted with 0.1 mL of elution buffer (50 mM MES, 2 mM EDTA, 150 mM NaCl,pH5.5) and contacted with 1 μL of 100 mg/mL trypsin to select for itscalcium dependency. After selection, selected phage clones wereconverted to IgG format.

Binding ability of converted IgG antibodies against human C5 wereassessed under two different conditions: association and dissociation at1.2 mM CaCl₂-pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM CaCl₂) andassociation at 1.2 mM CaCl₂-pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mMCaCl₂) and dissociation at 3 μM CaCl₂-pH 5.8 (20 mM MES, 150 mM NaCl, 3μM CaCl₂), at 30° C. using Octet RED384 system (Pall Life Sciences). 25clones of pH-Calcium dependent antigen binding clones were isolated. Thesensorgrams of these antibodies are shown in FIG. 38.

(22-4) Identification of Anti-C5 Bispecific Antibody

From the clones isolated in Example B-3, nine pH or calcium dependentanti-C5 antibody clones were selected for further analysis (CFP0008,0011, 0015, 0016, 0017, 0018, 0019, 0020, 0021). Some amino acidsubstitutions were introduced to the CFP0016 heavy chain variable regionby a method generally known to those of ordinary skill in the art toimprove properties of the antibodies like physicochemical properties.This CFP0016 variant, CFP0016H019, was used for further analysis insteadof CFP0016. The amino acid sequences of VH and VL regions of these nineantibodies are described in Table 34. In this table, names described inbrackets represent the abbreviated names.

TABLE 34 Clone Name and Amino Acid Sequence of Selected Antibodies CloneName VH Name VH SEQ ID VL Name VL SEQ ID CFP0008 (08) CFP0008H (08H) NO:209 CFP0008L (08L) NO: 210 CFP0011 (11) CFP0011H (11H) NO: 211 CFP0011L(11L) NO: 212 CFP0015 (15) CFP0015H (15H) NO: 213 CFP0015L (15L) NO: 214CFP0016H019 (16H019) CFP0016H019 (16H019) NO: 215 CFP0016L (16L) NO: 216CFP0017 (17) CFP0017H (17H) NO: 217 CFP0017L (17L) NO: 218 CFP0018 (18)CFP0018H (18H) NO: 219 CFP0018L (18L) NO: 220 CFP0019 (19) CFP0019H(19H) NO: 221 CFP0019L (19L) NO: 222 CFP0020 (20) CFP0020H (20H) NO: 223CFP0020L (20L) NO: 224 CFP0021 (21) CFP0021H (21H) NO: 225 CFP0021L(21L) NO: 226

The full-length genes having nucleotide sequences encoding antibodyheavy chain and light chain were synthesized and prepared by a methodgenerally known to those of ordinary skill in the art. Heavy chain andlight chain expression vectors were prepared by inserting the obtainedplasmid fragments into vectors for expression in mammalian cells. Theobtained expression vectors were sequenced by a method generally knownto those of ordinary skill in the art. For expression of antibodies, theprepared plasmids were transiently transfected to FreeStyle293-F cellline (Thermo Fisher Scientific). Purification from the conditioned mediaexpressing antibodies was conducted by a method generally known to thoseof ordinary skill in the art using rProtein A Sepharose Fast Flow (GEHealthcare).

(22-5) Generation and Characterization of pH Dependent Anti-05Bispecific Antibody

Bispecific antibodies, which recognize two different epitopes of C5,were generated by combination of CFP0020 and CFP0018. Bispecificantibody was prepared as IgG format having two different clones of Fabin each binding site of the antibody and was prepared using a methodgenerally known to those of ordinary skill in the art. In thisbispecific IgG antibody, two heavy chains comprise distinct heavy chainconstant regions (G1dP1, SEQ ID NO:227 and G1dN1, SEQ ID NO:228) fromeach other so as to efficiently form a heterodimer of the two heavychains. The anti-C5 bispecific antibody comprising the binding sites ofanti-05 MAb “X” and anti-05 MAb “Y” is represented as “X//Y”.

By introducing some amino acid substitutions into heavy chain and lightchain CDR by a method generally known to those of ordinary skill in theart, we obtained light chain communization variant of 20//18, which wenamed ‘optimized 20//18’ (consisted by two heavy chains:CFP0020H0261-G1dP1, SEQ ID NO:229 and CFP0018H0012-G1dN1, SEQ ID NO:230and common light chain: CFP0020L233-k0, SEQ ID NO:231).

The kinetics parameters of optimized 20//18 against recombinant human C5were assessed under two different conditions (e.g. (A) association anddissociation at pH 7.4 and (B) association at pH 7.4 and dissociation atpH 5.8), at 37° C. using BIACORE T200 instrument (GE Healthcare).Protein A/G (Pierce, Cat No. #21186) or anti-human IgG (Fc) antibody(within Human Antibody Capture Kit; GE Healthcare, Cat No. BR-1008-39)was immobilized onto a Series S CM4 (GE Healthcare, Cat No. BR-1005-34)by amine coupling method. Anti-05 antibodies were captured on animmobilized molecule, and then human C5 was injected. The runningbuffers used were ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2mM CaCl₂, 0.05% Tween 20). Kinetics parameters at both pH conditionswere determined by fitting the sensorgrams with 1:1 binding-RI (withoutbulk effect adjustment) model using BIACORE T200 Evaluation software,version 2.0 (GE Healthcare). Kinetic parameters, association rate (ka),dissociation rate (kd), and binding affinity (KD) at pH 7.4, anddissociation rate (kd) determined by only calculating the dissociationphase at each pH conditions, are described in Table 35. Optimized 20//18showed faster dissociation at pH 5.8 against human C5 compared withdissociation rate at pH 7.4.

TABLE 35 Kinetic Parameters of 20//18 Variants against Human C5 undertwo Different Conditions pH 7.4 pH 7.4 pH 5.8 ka kd KD kd (onlydissociation) optimized 3.17E+05 1.87E−04 5.89E−10 1.36E−04 4.84E−0220//18(22-6) Production of Antibodies with Increased pI by Modification ofAmino Acids in the Variable Region

The tested antibodies are summarized in Tables 36 and 37.

The heavy chain, CFP0020H0261-001-G1dP1 (also called 20H001, SEQ IDNO:232) was prepared by introducing pI-increasing substitution P41R/G44Rinto CFP0020H0261-G1dP1 (SEQ ID NO:229). Similarly, the heavy chain,CFP0018H0012-002-GldN1 (also called 18H002, SEQ ID NO:251) was preparedby introducing pI-increasing substitution T77R/E85R intoCFP0018H0012-G1dN1 (SEQ ID NO:230). Other heavy chain variants were alsoprepared by introducing respective substitutions represented in Table 36into CFP0020H0261-G1dP1 and CFP0018H0012-G1dN1 respectively, accordingto the method shown in Reference Example 1. The heavy chain variants ofboth CFP0020H0261-G1dP1 variants and CFP0018H0012-G1dN1 variants wereexpressed with CFP0020L233-k0 (SEQ ID NO:231) as light chain to obtainbi-specific antibody.

Similarly, we also evaluated the pI-increasing substitution in lightchain. The light chain, CFP0020L233-001-k0 (also called 20L233-001, SEQID NO:271) was prepared by introducing pI-increasing substitution G16Kinto CFP0020L233-k0. Other light chain variants were also prepared byintroducing respective substitutions represented in Table 37 intoCFP0020L233-k0 according to the method shown in Reference Example 1. Allthe light chain variants were expressed with CFP0020H0261-G1dP1 andCFP0018H0012-G1dN1 as heavy chain to obtain bi-specific antibody.

TABLE 36 Heavy Chain Variants of CFP0020H0261-001-G1dP1 andCFP0018H0012-001-G1dN1 evaluated in this Example Sample Name MutationMutation (Heavy Chain 1/Heavy (Heavy (Heavy Imaging BIACORE Chain2/Light Chain) Variant Chain 1) Chain 2) fold fold CFP0020H0261-G1dP1/original 1.00 1.00 CFP0018H0012-G1dN1/ Ab2 CFP0020L233-k0CFP0020H0261-001-G1dP1/ 20H001/ P41R/G44R T77R/E85G 2.27 0.91CFP0018H0012-002-G1dN1/ 18H002 CFP0020L233-k0 CFP0020H0261-002-G1dP1/20H002/ Q77R/A85R T77R/E85G 2.46 1.09 CFP0018H0012-002-G1dNl/ 18H002CFP0020L233-k0 CFP0020H0261-003-G1dP1/ 20H003/ L18R G8R 1.69 0.97CFP0018H0012-003-G1dN1/ 18H003 CFP0020L233-k0 CFP0020H0261-005-G1dP1/20H005/ S15R G15R 1.36 0.96 CFP0018H0012-005-G1dN1/ 18H005CFP0020L233-k0 CFP0020H0261-008-G1dP1/ 20H008/ G32R Y32R 0.00 0.20CFP0018H0012-008-G1dN1/ 18H008 CFP0020L233-k0 CFP0020H0261-009-G1dP1/20H009/ Q39K Q39K 1.45 1.01 CFP0018H0012-009-G1dN1/ 18H009CFP0020L233-k0 CFP0020H0261-013-G1dP1/ 20H013/ L63R F63R 2.64 1.52CFP0018H0012-013-G1dN1/ 18H013 CFP0020L233-k0 CFP0020H0261-G1dP1/ 20H/ —Q64K 0.86 0.80 CFP0018H0012-014-G1dN1/ 18H014 CFP0020L233-k0CFP0020H0261-G1dP1/ 20H/ — F63R/Q64K 1.90 1.08 CFP0018H0012-016-G1dN1/18H016 CFP0020L233-k0 CFP0020H0261-018-G1dP1/ 20H018/ Q77R T77R 2.800.96 CFP0018H0012-018-G1dN1/ 18H018 CFP0020L233-k0CFP0020H0261-019-G1dP1/ 20H019/ L82K L82K 2.47 1.61CFP0018H0012-019-G1dN1/ 18H019 CFP0020L233-k0 CFP0020H0261-020-G1dP1/20H020/ S82aN/S82bR S82aN/S82bR 1.45 0.93 CFP0018H0012-020-G1dN1/ 18H020CFP0020L233-k0 CFP0020H0261-021-G1dP1/ 20H021/ S82aG/S82bR S82aG/S82bR0.74 0.85 CFP0018H0012-021-G1dN1/ 18H021 CFP0020L233-k0CFP0020H0261-022-G1dP1/ 20H022/ S82bR S82bR 1.25 0.86CFP0018H0012-022-G1dN1/ 18H022 CFP0020L233-k0 CFP0020H0261-023-G1dP1/20H023/ V82cR L82cR 0.58 0.52 CFP0018H0012-023-G1dN1/ 18H023CFP0020L233-k0 CFP0020H0261-G1dP1/ 20H/ — E85G 0.99 0.72CFP8810H0012-024-G1dN1/ 18H024 CFP0020L233-k0 CFP0020H0261-025-G1dP1/20H025/ D86G D86G 0.68 0.76 CFP0018H0012-025-G1dN1/ 18H025CFP0020L233-k0 CFP0020H0261-026-G1dP1/ 20H026/ A93K A93K 0.01 0.23CFP0018H0012-026-G1dN1/ 18H026 CFP0020L233-k0 CFP0020H0261-G1dP1/ 20H/ —Q105R 1.22 0.79 CFP0018H0012-027-G1dN1/ 18H027 CFP0020L233-k0CFP0020H0261-032-G1dP1/ 20H032/ L82K/S82bR L82K/S82bR 1.47 2.25CFP0018H0012-032-G1dN1/ 18H032 CFP0020L233-k0 CFP0020H0261-035-G1dP1/20H035/ S82bR/T83R — 1.03 1.15 CFP0018H0012-G1dN1/ 18H CFP0020L233-k0CFP0020H0261-036-G1dP1/ 20H036/ T83R — 2.02 1.05 CFP0018H0012-G1dN1/ 18HCFP0020L233-k0 CFP0020H0261-037-G1dP1/ 20H037/ V71R/A85G A71R/E85G 0.340.49 CFP0018H0012-037-G1dN1/ 18H037 CFP0020L233-k0

TABLE 37 Light Chain Variants of CFP0020L233 evaluated in this ExampleImaging BIACORE Sample Name Variant Mutation fold foldCFP0020H0261-G1dP1/ original 1.00 1.00 CFP0018H0012-G1dN1/ Ab2CFP0020L233-k0 CFP0020H0261-G1dP1/ 20L233- G16K 2.20 1.59CFP0018H0012-G1dN1/ 001 CFP0020L233-001-k0 CFP0020H0261-G1dP1/ 20L233-Q27R 1.08 0.92 CFP0018H0012-G1dN1/ 002 CFP0020L233-002-k0CFP0020H0261-G1dP1/ 20L233- A25R/S26R 0.38 0.32 CFP0018H0012-G1dN1/ 003CFP0020L233-003-k0 CFP0020H0261-G1dP1/ 20L233- S52K/S56K 1.24 0.75CFP0018H0012-G1dN1/ 004 CFP0020L233-004-k0 CFP0020H0261-G1dP1/ 20L233-T74K/S77R 3.32 1.83 CFP0018H0012-G1dN1/ 005 CFP0020L233-005-k0CFP0020H0261-G1dP1/ 20L233- S76R/Q79K 4.85 1.89 CFP0018H0012-G1dN1/ 006CFP0020L233-006-k0 CFP0020H0261-G1dP1/ 20L233- Q27K 1.18 0.95CFP0018H0012-G1dN1/ 007 CFP0020L233-007-k0 CFP0020H0261-G1dP1/ 20L233-A25K/S26K 0.29 0.29 CFP0018H0012-G1dN1/ 008 CFP0020L233-008-k0CFP0020H0261-G1dP1/ 20L233- Q37R 0.99 0.78 CFP0018H0012-G1dN1/ 009CFP0020L233-009-k0 CFP0020H0261-G1dP1/ 20L233- G41R 1.77 1.01CFP0018H0012-G1dN1/ 010 CFP0020L233-010-k0 CFP0020H0261-G1dP1/ 20L233-L46R/Y49K 0.00 0.14 CFP0018H0012-G1dN1/ 011 CFP0020L233-011-k0CFP0020H0261-G1dP1/ 20L233- S52R/S56R 1.02 0.68 CFP0018H0012-G1dN1/ 012CFP0020L233-012-k0 CFP0020H0261-G1dP1/ 20L233- S65R/T69R 1.24 No dataCFP0018H0012-G1dN1/ 013 CFP0020L233-013-k0 CFP0020H0261-G1dP1/ 20L233-G41R/T74K/S77R 6.58 1.96 CFP0018H0012-G1dN1/ 016 CFP0020L233-016-k0CFP0020H0261-G1dP1/ 20L233- L46R/Y49K/T74K/S77R 0.00 0.18CFP0018H0012-G1dN1/ 017 CFP0020L233-017-k0 CFP0020H0261-G1dP1/ 20L233-S52R/S56R/T74K/S77R 46.97 1.43 CFP0018H0012-G1dN1/ 018CFP0020L233-018-k0 CFP0020H0261-G1dP1/ 20L233- S65R/T69R/T74K/S77R 53.257.72 CFP0018H0012-G1dN1/ 019 CFP0020L233-019-k0 CFP0020H0261-G1dP1/20L233- Q27R/S76R/Q79K 20.90 1.77 CFP0018H0012-G1dN1/ 021CFP0020L233-021-k0 CFP0020H0261-G1dP1/ 20L233- G41R/S76R/Q79K 27.3 2.04CFP0018H0012-G1dN1/ 022 CFP0020L233-022-k0 CFP0020H0261-G1dP1/ 20L233-L46R/Y49K/S76R/Q79K 0.2 0.18 CFP0018H0012-G1dN1/ 023 CFP0020L233-023-k0CFP0020H0261-G1dP1/ 20L233- S52R/S56R/S76R/Q79K 114.7 2.16CFP0018H0012-G1dN1/ 024 CFP0020L233-024-k0 CFP0020H0261-G1dP1/ 20L233-S65R/T69R/S76R/Q79K 75.6 3.18 CFP0018H0012-G1dN1/ 025 CFP0020L233-025-k0CFP0020H0261-G1dP1/ 20L233- Q27R/G41R/T74K/S77R 4.1 2.24CFP0018H0012-G1dN1/ 027 CFP0020L233-027-k0 CFP0020H0261-G1dP1/ 20L233-G41R/S52R/S56R/T74K/S77R 18.1 1.94 CFP0018H0012-G1dN1/ 028CFP0020L233-028-k0 CFP0020H0261-G1dP1/ 20L233- G41R/S65R/T69R/T74K/S77R28.8 9.63 CFP0018H0012-G1dN1/ 029 CFP0020L233-029-k0 CFP0020H0261-G1dP1/20L233- Q27R/S52R/S56R/T74K/S77R 21.0 1.78 CFP0018H0012-G1dN1/ 031CFP0020L233-031-k0 CFP0020H0261-G1dP1/ 20L233-S52R/S56R/S65R/T69R/T74K/S77R 64.1 11.09  CFP0018H0012-G1dN1/ 032CFP0020L233-032-k0 CFP0020H0261-G1dP1/ 20L233- Q27R/S65R/T69R/T74K/S77R30.5 8.61 CFP0018H0012-G1dN1/ 034 CFP0020L233-034-k0 CFP0020H0261-G1dP1/20L233- Q27R/G41R/S76R/Q79K 14.5 2.45 CFP0018H0012-G1dN1/ 036CFP0020L233-036-k0 CFP0020H0261-G1dP1/ 20L233- G41R/S52R/S56R/S76R/Q79K53.0 2.43 CFP0018H0012-G1dN1/ 037 CFP0020L233-037-k0 CFP0020H0261-G1dP1/20L233- G41R/S65R/T69R/S76R/Q79K 45.9 4.24 CFP0018H0012-G1dN1/ 038CFP0020L233-038-k0 CFP0020H0261-G1dP1/ 20L233- Q27R/S52R/S56R/S76R/Q79K61.6 2.32 CFP0018H0012-G1dN1/ 040 CFP0020L233-040-k0 CFP0020H0261-G1dP1/20L233- S52R/S56R/S65R/T69R/S76R/Q79K 96.2 5.28 CFP0018H0012-G1dN1/ 041CFP0020L233-041-k0 CFP0020H0261-G1dP1/ 20L233- Q27R/S65R/T69R/S76R/Q79K49.3 3.51 CFP0018H0012-G1dN1/ 043 CFP0020L233-043-k0 CFP0020H0261-G1dP1/20L233- S76R 1.61 1.25 CFP0018H0012-G1dN1/ 044 CFP0020L233-044-k0CFP0020H0261-G1dP1/ 20L233- S65R/Q79K 3.66 1.46 CFP0018H0012-G1dN1/ 045CFP0020L233-045-k0(22-7) Human FcγRIIb-Binding Assay by BIACORE Using pI-IncreasedVariants

Regarding the produced Fc region variant-containing antibodies, bindingassays between soluble hFcγRIIb and antigen-antibody complexes wereperformed using BIACORE T200 (GE Healthcare). Soluble hFcγRIIb wasproduced in the form of a His-tagged molecule by a method known in theart. An appropriate amount of an anti-His antibody was fixed onto Sensorchip CM5 (GE Healthcare) by the amine coupling method using a Hiscapture kit (GE Healthcare) to capture hFcγRIIb. Next, anantibody-antigen complex and a running buffer (as a reference solution)were injected, and interaction was allowed to take place with thehFcγRIIb captured onto the sensor chip. 20 mMN-(2-Acetamido)-2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl₂,and 0.05% (w/v) Tween 20 at pH 7.4 was used as the running buffer, andthe respective buffer was also used to dilute the soluble hFcγRIIb. Toregenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. Allmeasurements were carried out at 25° C. Analyses were performed based onbinding (RU) calculated from sensorgrams obtained by the measurements,and relative values when the binding amount ofCFP0020H0261-G1dP1/CFP0018H0012-G1dN1/CFP0020L233-k0 (original Ab2) wasdefined as 1.00 are shown. To calculate the parameters, the BIACORE T100Evaluation Software (GE Healthcare) was used.

The SPR analysis results are summarized in Tables 36 and 37. A fewvariants were shown to have enhanced binding toward hFcγRIIb fixed onthe BIACORE sensor chip. Here, about 1.2 fold or more of the binding tohFcγRIIb of the variants compared to the binding to hFcγRIIb of originalAb2 was considered to have strong charge effect on binding of anantibody to hFcγRIIb on the sensor chip.

Among the pI-increased heavy chain variants, the antibody with L63R,F63R, L82K or S82bR substitutions (according to Kabat numbering) showedhigher binding to hFcγRIIb. The single amino acid substitution or acombination of these substitutions in heavy chain is supposed to havestrong charge effect on binding to hFcγRIIb on the sensor chip. Thus,one or more of positions that are expected to show an effect ofaccelerating the speed or rate of uptake into cells in vivo byintroducing the pI-increasing modification into the heavy chain variableregion(s) of an antibody can include, for example, position 63, 82 or82b according to Kabat numbering. An amino acid substitution introducedat such position(s) can be arginine or lysine.

In the pI-increased light chain variants, the antibody with G16K, Q27R,G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitutions(according to Kabat numbering) showed higher binding to hFcγRIIb. Thesingle amino acid substitution or a combination of these substitutionsin light chain is supposed to have strong charge effect on binding tohuman FcγRIIb on the sensor chip. Thus, one or more of positions thatare expected to show an effect of accelerating the speed or rate ofuptake into cells in vivo by introducing the pI-increasing modificationinto the light chain variable region of an antibody can include, forexample, positions 16, 27, 41, 52, 56, 65, 69, 74, 76, 77 or 79,according to Kabat numbering. An amino acid substitution introduced atsuch position(s) can be arginine or lysine. The variants with four ormore amino acid substitutions tended to show stronger charge effect thanthose variants with lesser amino acid substitutions.

(22-8) Cellular Uptake of pI-Increased Fab Region Variant-ContainingAntibodies

To evaluate the rate of intracellular uptake into an hFcγRIIb-expressingcell line using the produced Fab region variant-containing antibodies,the following assay was performed.

An MDCK (Madin-Darby canine kidney) cell line that constitutivelyexpresses hFcγRIIb was produced by known methods. Using these cells,intracellular uptake of antigen-antibody complexes was evaluated.Specifically, Alexa555 (Life Technlogies) was used to label human C5according to an established protocol, and antigen-antibody complexeswere formed in a culture solution with the antibody concentration being10 mg/mL and the antigen concentration being 10 mg/mL. The culturesolution containing the antigen-antibody complexes was added to cultureplates of the above-mentioned MDCK cells which constitutively expresshFcγRIIb and incubated for one hour, and then the fluorescence intensityof the antigen taken up into the cells was quantified using InCellAnalyzer 6000 (GE healthcare). The amount of antigen taken up waspresented as relative values to the original Ab2 value which is taken as1.00.

The quantification results of cellular uptake were summarized in Tables36 and 37. Strong fluorescence derived from the antigen in the cells wasobserved in several heavy chain and light chain variants. Here, about1.5 fold or more of the fluorescence intensity of the antigen taken upinto the cells of the variants compared to the fluorescence intensity oforiginal Ab2 was considered to have strong charge effect on an antigentaken up into the cells.

Among the pI-increased heavy chain variants, the antibody with G8R,L18R, Q39K, P41R, G44R, L63R, F63R, Q64K, Q77R, T77R, L82K, S82aN,S82bR, T83R, A85R or E85G substitution(s) (according to Kabat numbering)showed stronger antigen uptake into the cells. The single amino acidsubstitution or a combination of these substitutions in heavy chain issupposed to have strong charge effect on antigen antibody complex uptakeinto the cells. Thus, one or more of positions that are expected tocause uptake of an antigen-antibody complex into cells more quickly ormore frequently by introducing the pI-increasing modification into theheavy chain variable region(s) of an antibody can include, for example,positions 8, 18, 39, 41, 44, 63, 64, 77, 82, 82a, 82b, 83, or 85,according to Kabat numbering. An amino acid substitution introduced atsuch position(s) can be asparagine, glycine, serine, arginine or lysine,and preferably arginine or lysine.

In the pI-increased light chain variants, the antibody with G16K, Q27R,S27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79Ksubstitutions (according to Kabat numbering) showed stronger antigenuptake into the cells. The single amino acid substitution or acombination of these substitutions in light chain is supposed to havestrong charge effect on antigen antibody complex uptake into the cells.The variants with four or more amino acid substitutions tended to showstronger charge effect than those variants with lesser amino acidsubstitutions. As shown in Example 21, (21-3), the combination of 42Kand 76R substitution is effective in IgE antibody. In the case of C5antibody, however, the amino acid of Kabat numbering 42 is alreadylysine, so we can observe the charge effect of 42K/76R by the singlesubstitution of 76R. The fact that the variant with 76R substitution hadstrong charge effect also in C5 antibody shows the combination of42K/76R has strong charge effect regardless of antigen. Thus, one ormore of positions that are expected to cause uptake of anantigen-antibody complex into cells more quickly or more frequently byintroducing the pI-increasing modification into the light chain variableregion(s) of an antibody can include, for example, positions 16, 27, 41,52, 56, 65, 69, 74, 76, 77 or 79, according to Kabat numbering. An aminoacid substitution introduced at such position(s) can be arginine orlysine.

(22-9) Evaluation of Clearance of C5 in Mouse Co-Injection Model

Some anti-C5 bispecific antibodies (original Ab2, 20L233-005, 20L233-006and 20L233-009) were tested in mice co-injection model to evaluate theirability to accelerate the clearance of C5 from plasma. In co-injectionmodel, C57BL6J mice (Jackson Laboratories) were administered by singlei.v. injection with C5 pre-mixed with the anti-C5 bispecific antibody,respectively. All groups received 0.1 mg/kg C5 with 1.0 mg/kg of anti-C5bispecific antibodies. Total C5 plasma concentration was determined byanti-C5 ECLIA. First, anti-human C5 mouse IgG was dispensed into an ECLplate, and left for overnight at 5° C. to prepare an anti-human C5 mouseIgG-immobilized plate. Samples for standard curve and samples were mixedwith an anti-human C5 rabbit IgG. These samples were added into theanti-human C5 mouse IgG-immobilized plate, and left for one hour at roomtemperature. Then, these samples were reacted with HRP-conjugatedanti-rabbit IgG (Jackson Immuno Research). After the plate was incubatedfor one hour at room temperature, a sulfo-tag conjugated anti-HRP wereadded. ECL signal was read with Sector Imager 2400 (Meso Scalediscovery). The concentration of human C5 was calculated from the ECLsignal in the standard curve using SOFTmax PRO (Molecular Devices). FIG.39 describes the C5 plasma concentration time profile in C57BL6J mice.

Compared to original Ab2, all of the bispecific antibodies withpI-increased substitution(s) tested in this study demonstrated rapid C5clearance from plasma. Therefore, amino acid substitution(s) onT74K/S77R, S76R/Q79K and Q37R in light chain are suggested to accelerateelimination of C5-antibody immune complex also in vivo. Furthermore, C5elimination of 20L233-005 and 20L233-006 was faster than that of20L233-009, that was consistent with in vitro imaging and BIACOREanalysis. These results suggest that even the position(s) which seemsunlikely to contribute for clearance of an antigen from plasma in vivo,examined under either the in vitro system using the fluorescenceintensity by InCell Analyzer 6000 or the in vitro BIACORE systemdescribed above, can be found out to contribute for that by using themore sensitive in vivo system. These results also suggest that forspeculating an evaluation of clearance of an antigen from plasma invivo, the sensitivity of the in vitro system using the fluorescenceintensity by InCell Analyzer 6000 described above may be higher thanthat of the in vitro BIACORE system described above.

Example 23 Evaluation of Clearance of IgE from Plasma Using pI-IncreasedFc Variants

To enhance the clearance of human IgE or human C5, pI-increasedsubstitutions in the Fc portion of antibodies were evaluated using pHdependent antibodies. The method of adding amino acid substitutions tothe antibody constant region to increase pI is not particularly limited,but for example, it can be performed by the method described inWO2014/145159.

(23-1) Production of Antibodies with Increased-pI by a Single Amino AcidModification in the Constant Region

The tested antibodies are summarized in Table 38. The heavy chain,Ab1H-P1394m (SEQ ID NO:307) was prepared by introducing a pI-increasingsubstitution Q311K into Ab1H. Other heavy chain variants were alsoprepared by introducing respective substitutions represented in Table 38into Ab1H according to the method shown in Reference Example 1. All theheavy chain variants were expressed with Ab1L as light chain.

TABLE 38 Antibody Name (Heavy Chain/ Imaging BIACORE Light Chain)Variant Mutation fold fold Ab1H/Ab1L original Ab1 — 1.00 1.00Ab1H-P1394m/Ab1L P1394m Q311K 1.31 1.18 Ab1H-P1398m/Ab1L P1398m D413K3.45 1.23 Ab1H-P1466m/Ab1L P1466m Q311R 1.90 1.22 Ab1H-P1468m/Ab1LP1468m N315R 1.42 1.13 Ab1H-P1469m/Ab1L P1469m N315K 1.93 1.13Ab1H-P1470m/Ab1L P1470m N384R 1.50 1.19 Ab1H-P1471m/Ab1L P1471m N384K0.71 1.19 Ab1H-P1480m/Ab1L P1480m Q342R 1.08 1.03 Ab1H-P1481m/Ab1LP1481m Q342K 1.83 1.08 Ab1H-P1482m/Ab1L P1482m P343R 4.90 1.46Ab1H-P1483m/Ab1L P1483m P343K 1.99 1.02 Ab1H-P1512m/Ab1L P1512m D401R2.98 1.24 Ab1H-P1513m/Ab1L P1513m D401K 2.57 1.21 Ab1H-P1514m/Ab1LP1514m G402R 1.22 1.20 Ab1H-P1515m/Ab1L P1515m G402K 0.93 1.19Ab1H-P1653m/Ab1L P1653m D413R 3.96 0.79(23-2) Human FcγRIIb-Binding Assay by BIACORE Using pI-Increased FcRegion Variant-Containing Antibodies

To evaluate the charge effect on FcRγRIIb-binding of antigen-antibodycomplex formed by using the antibodies described in Table 38,FcRγRIIb-binding assay was performed in a similar manner with thosedescribed in Example 21, (21-2). Assay results are shown in Table 38.Here, about 1.2 fold or more of the binding to hFcγRIIb of the variantscompared to the binding to hFcγRIIb of original Ab1 was considered tohave strong charge effect on binding of an antibody to hFcγRIIb on thesensor chip.

Among the pI-increased variants with a single amino acid substitutionfrom original Ab1, the antigen-antibody complex made by several variantssuch as P1398m, P1466m, P1482m, P1512m, P1513m, and P1514m showedhighest binding to hFcγRIIb. The single amino acid substitution onD413K, Q311R, P343R, D401R, D401K, G402R, Q311K, N384R, N384K, or G402Kis supposed to have strong charge effect on binding to hFcγRIIb on thesensor chip. Thus, a single position that is expected to show an effectof accelerating the speed or rate of uptake into cells in vivo byintroducing the pI-increasing modification into the constant or Fcregion of an antibody can include, for example, positions 311, 343, 384,401, 402, or 413, according to EU numbering. An amino acid substitutionintroduced at such position can be arginine or lysine.

(23-3) Cellular Uptake of pI-Increased Fc Region Variant-ContainingAntibodies

To evaluate the intracellular uptake of antigen-antibody complex formedby the antibodies described in Table 38, cell imaging assay wasperformed in a similar manner with those described in Example 21,(21-3). Assay results are shown in Table 38. Here, about 1.5 fold ormore of the fluorescence intensity of the antigen taken up into thecells of the variants compared to the fluorescence intensity of originalAb1 was considered to have strong charge effect on an antigen taken upinto the cells.

Among the pI-increased variants with a single amino acid substitutionfrom original Ab1, the antigen-antibody complex made by several variantssuch as P1398m, P1466m, P1469m, P1470m, P1481m, P1482m, P1483m, P1512m,P1513m and P1653m showed stronger antigen uptake into the cells. Thesingle amino acid substitution on D413K, Q311R, N315K, N384R, Q342K,P343R, P343K, D401R, D401K or D413R is supposed to have strong chargeeffect on antigen antibody complex uptake into the cells. Thus, a singleposition that is expected to cause uptake of an antigen-antibody complexinto cells more quickly or more frequently by introducing thepI-increasing modification into the constant or Fc region of an antibodycan include, for example, positions 311, 315, 342, 343, 384, 401, or413, according to EU numbering. An amino acid substitution introduced atsuch position can be arginine or lysine.

(23-4) Evaluation of Clearance of Human IgE in Mouse Co-Injection Model

Some anti-IgE antibodies with pH-dependent antigen-binding (originalAb1, P1466m, P1469m, P1470m, P1480m, P1482m, P1512m, P1653m) were testedin mice co-injection model to evaluate their ability to accelerate theclearance of IgE from plasma. The assays were performed in a similar waywith Example 21, (21-4). FIG. 40 describes the plasma concentration timeprofile in C57BL6J mice.

After administration of high pI variants (only a single amino acidsubstitution) with pH-dependent antigen-binding, the plasma total IgEconcentration was lower than that of original Ab1 except for P1480m.P1480m, which showed weak efficacy the both in vitro studies, did notaccelerate elimination of IgE. Furthermore, the plasma total IgEconcentration in mice treated with high pI variant without pH-dependentantigen-binding was significantly higher than that of high pI variantwith pH-dependent antigen-binding (data not shown). These resultsindicate that the cellular uptake of antigen-antibody immune complexincrease by introducing the pI-increasing modification. The antigenuptaken into the cells in complex with a pH-dependent antigen-bindingantibody could release from antibody inside endosome effectively,resulted in accelerated elimination of IgE. These results suggest thateven the substituted position which seems unlikely to contribute forclearance of an antigen from plasma in vivo, examined under the in vitroBIACORE system described above, can be found out to contribute for thatby using the more sensitive in vivo system. These results also suggestthat for speculating an evaluation of clearance of an antigen fromplasma in vivo, the sensitivity of the in vitro system using thefluorescence intensity by InCell Analyzer 6000 described above may behigher than that of the in vitro BIACORE system described above.

Reference Example 1 Construction of Expression Vectors of AminoAcid-Substituted IgG Antibodies

Mutants were prepared using the QuikChange Site-Directed Mutagenesis Kit(Stratagene) by the method described in the appended instruction manual.Plasmid fragments containing the mutants were inserted into animal cellexpression vectors to construct desired H-chain and L-chain expressionvectors. The nucleotide sequences of the obtained expression vectorswere determined by methods known in the art.

Reference Example 2 Expression and Purification of IgG Antibodies

Antibodies were expressed using the following method. The humanembryonic kidney cancer cell-derived HEK293H cell line (Invitrogen) wassuspended in DMEM medium (Invitrogen) supplemented with 10% Fetal BovineSerum (Invitrogen). The cells were plated at 10 mL per dish in dishesfor adherent cells (10 cm in diameter; CORNING) at a cell density of 5to 6×10⁵ cells/mL and cultured in a CO₂ incubator (37° C., 5% CO₂) forone day. Then, the medium was removed by aspiration, and 6.9 mL ofCHO-S-SFM-II medium (Invitrogen) was added. The prepared plasmid wasintroduced into the cells by the lipofection method. The resultingculture supernatants were collected, centrifuged (approximately 2,000 g,5 minutes, room temperature) to remove cells, and sterilized byfiltering through the 0.22-μm filter MILLEX (registered trademark)-GV(Millipore) to obtain supernatants. Antibodies were purified from theobtained culture supernatants by methods known in the art using therProtein A Sepharose™ Fast Flow (Amersham Biosciences). To determine theconcentration of the purified antibody, absorbance was measured at 280nm using a spectrophotometer. Antibody concentrations were calculatedfrom the determined values using an absorbance coefficient calculated bythe method described in Pace et al., Protein Science 4:2411-2423 (1995).

Reference Example 3 Preparation of a Soluble Human IL-6 Receptor

A recombinant soluble human IL-6 receptor, which is an antigen, wasprepared in the manner described below. A CHO cell line thatconstitutively expresses soluble human IL-6 receptor composed of anamino acid sequence of the 1st to 357th amino acid from the N terminusas reported in Mullberg et al., J. Immunol. 152:4958-4968 (1994) wasconstructed using a method known in the art. Soluble human IL-6 receptorwas expressed by culturing this CHO line. Soluble human IL-6 receptorwas purified from the culture supernatant of the obtained CHO line intwo steps: Blue Sepharose 6 FF column chromatography and a gelfiltration column chromatography. The fraction that was eluted as themain peak in the final step was used as the final purified product.

What is claimed is:
 1. An isolated anti-IL-8 antibody which comprises(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:67, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:73, (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:74, (d) HVR-LIcomprising the amino acid sequence of SEQ ID NO:70, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:75, and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:76.
 2. The anti-IL-8antibody of claim 1, which is an IgG antibody.
 3. The anti-IL-8 antibodyof claim 2, wherein the IgG antibody is IgG1, IgG2, IgG3 or IgG4.
 4. Theanti-IL-8 antibody of claim 3, wherein the IgG antibody is IgG1.
 5. Theanti-IL-8 antibody of claim 1, which comprises one or more amino acidsubstitutions selected from the group consisting of: L235R, G236R,S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E,according to EU numbering.
 6. The anti-IL-8 antibody of claim 5, whichcomprises amino acid substitution N434A according to EU numbering. 7.The anti-IL-8 antibody of claim 6, which comprises amino acidsubstitutions of L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, andS440E, according to EU numbering.
 8. The anti-IL-8 antibody of claim 6,which comprises amino acid substitutions of L235R, G236R, A327G, A330S,P331S, M428L, N434A, Y436T, Q438R, and S440E, according to EU numbering.9. A pharmaceutical composition comprising the anti-IL-8 antibody ofclaim 1 and a pharmaceutically acceptable carrier.
 10. A pharmaceuticalcomposition comprising the anti-IL-8 antibody of claim 5 and apharmaceutically acceptable carrier.
 11. A pharmaceutical compositioncomprising the anti-IL-8 antibody of claim 6 and a pharmaceuticallyacceptable carrier.
 12. A pharmaceutical composition comprising theanti-IL-8 antibody of claim 7 and a pharmaceutically acceptable carrier.13. A pharmaceutical composition comprising the anti-IL-8 antibody ofclaim 8 and a pharmaceutically acceptable carrier.
 14. An isolatedanti-IL-8 antibody which comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:78 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:79. 15.The anti-IL-8 antibody of claim 14, which is an IgG antibody.
 16. Theanti-IL-8 antibody of claim 15, wherein the IgG antibody is IgG1, IgG2,IgG3 or IgG4.
 17. The anti-IL-8 antibody of claim 16, wherein the IgGantibody is IgG1.
 18. The anti-IL-8 antibody of claim 14, whichcomprises one or more amino acid substitutions selected from the groupconsisting of: L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A,Y436T, Q438R, and S440E, according to EU numbering.
 19. The anti-IL-8antibody of claim 18, which comprises amino acid substitution N434Aaccording to EU numbering.
 20. The anti-IL-8 antibody of claim 19, whichcomprises amino acid substitutions of L235R, G236R, S239K, M428L, N434A,Y436T, Q438R, and S440E, according to EU numbering.
 21. The anti-IL-8antibody of claim 19, which comprises amino acid substitutions of L235R,G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E,according to EU numbering.
 22. The anti-IL-8 antibody of claim 14, whichcomprises a heavy chain comprising an amino acid sequence selected fromthe group consisting of: (a) SEQ ID NO:80; (b) SEQ ID NO:81; (c) SEQ IDNO:92; and (d) SEQ ID NO:93.
 23. The anti-IL-8 antibody of claim 14,which comprises a light chain comprising the amino acid sequence of SEQID NO:82.
 24. The anti-IL-8 antibody of claim 14, which comprises alight chain comprising the amino acid sequence of SEQ ID NO:82 and aheavy chain comprising an amino acid sequence selected from the groupconsisting of: (a) SEQ ID NO:80; (b) SEQ ID NO:81; (c) SEQ ID NO:92; and(d) SEQ ID NO:93.
 25. The anti-IL-8 antibody of claim 24, whichcomprises a light chain comprising the amino acid sequence of SEQ IDNO:82 and a heavy chain comprising the amino acid sequence of SEQ IDNO:80.
 26. The anti-IL-8 antibody of claim 24, which comprises a lightchain comprising the amino acid sequence of SEQ ID NO:82 and a heavychain comprising the amino acid sequence of SEQ ID NO:81.
 27. Apharmaceutical composition comprising the anti-IL-8 antibody of claim 14and a pharmaceutically acceptable carrier.
 28. A pharmaceuticalcomposition comprising the anti-IL-8 antibody of claim 18 and apharmaceutically acceptable carrier.
 29. A pharmaceutical compositioncomprising the anti-IL-8 antibody of claim 19 and a pharmaceuticallyacceptable carrier.
 30. A pharmaceutical composition comprising theanti-IL-8 antibody of claim 20 and a pharmaceutically acceptablecarrier.
 31. A pharmaceutical composition comprising the anti-IL-8antibody of claim 21 and a pharmaceutically acceptable carrier.
 32. Apharmaceutical composition comprising the anti-IL-8 antibody of claim 22and a pharmaceutically acceptable carrier.
 33. A pharmaceuticalcomposition comprising the anti-IL-8 antibody of claim 23 and apharmaceutically acceptable carrier.
 34. A pharmaceutical compositioncomprising the anti-IL-8 antibody of claim 24 and a pharmaceuticallyacceptable carrier.
 35. A pharmaceutical composition comprising theanti-IL-8 antibody of claim 25 and a pharmaceutically acceptablecarrier.
 36. A pharmaceutical composition comprising the anti-IL-8antibody of claim 26 and a pharmaceutically acceptable carrier.